Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices

ABSTRACT

Methods for the automated template-free synthesis of user-defined sequence controlled biopolymers using microfluidic devices are described. The methods facilitate simultaneous synthesis of up to thousands of uniquely addressed biopolymers from the controlled movement and combination of regents as fluid droplets using microfluidic and EWOD-based systems. In some forms, biopolymers including nucleic acids, peptides, carbohydrates, and lipids are synthesized from step-wise assembly of building blocks based on a user-defined sequence of droplet movements. In some forms, the methods synthesize uniquely addressed nucleic acids of up to 1,000 nucleotides in length. Methods for adding, removing and changing barcodes on biopolymers are also provided. Biopolymers synthesized according to the methods, and libraries and databases thereof are also described. Modified biopolymers, including chemically modified nucleotides and biopolymers conjugated to other molecules are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ApplicationNo. 62/521,612 filed Jun. 19, 2017, the contents of which isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.N00014-16-121953 and Grant No. N00014-17-1-2609 awarded by the Office ofNaval Research, under Grant No. DE-SC0001088 awarded by the U.S.Department of Energy Office of Basic Energy Sciences, and under GrantNo. CCF-1564025 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jun. 19, 2018, as a text file named“MIT_19620_ST25.txt,” created on Jun. 19, 2018, and having a size of5,000 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to the automated de novo synthesis ofnucleic acids and other biopolymers, and in particular to the use ofelectrowetting on dielectric, microfluidic, and liquid handlingtechnology for high-throughput and dynamic production of biopolymers.

BACKGROUND OF THE INVENTION

DNA synthesis is often viewed as the next generation problem followingon the successes of DNA sequencing. This global vision is embodied byrecent efforts such as Human Genome Write where the goal is synthesis ofa synthetic human genome. The need for synthesis of long strands of DNA(i.e., greater than 2,000 bases) is additionally shown by Yeast 2.0,minimal cell projects, and is a fundamental enabling technology ofsynthetic biology.

Two major approaches to DNA synthesis are phosphoramidite (chemical)synthesis and enzymatic synthesis. The synthesis of oligonucleotides(oligos) was first achieved in the 1950s by Todd, Khorana and co-workersusing solution-based synthesis. (Todd, J. Chem. Soc., pp. 2632-2638(1955); Khorana, J. Am. Chem. Soc., 79 (4): pp. 1002-1003 (1957)). Inthe 1980s Caruthers developed oligonucleotide synthesis on insolublesupport using phosphoramidite synthons, which is currently thepredominant method to synthesize oligonucleotide strands (CaruthersTetrahedr. Lett. 22:1859-1862(1981)). The first step to synthesizingoligonucleotides using phosphoramidite precursors is to cleave the5′-dimethoxytrityl protecting group from a 2′-deoxynucleoside covalentlyattached to controlled pore glass (the insoluble support). A protected2′-deoxynucleoside-3′-phosphoramidite is then added to the support withtetrazole, which activates the added phosphoramidite. The formation ofthe covalent phosphite triester linkage occurs within 30 s. Next, anacetylation step using acetic anhydride with pyridine caps any unreacted2′-deoxynucleoside, and removes phosphite adducts from the nucleobases.Finally, an oxidation step with iodine converts the phosphite linkage toa phosphate group. This cycle is repeated until the desired oligosequence is synthesized, and then the oligo is cleaved from the solidsupport. Simultaneous synthesis of 96-768 oligonucleotides using thiscolumn-based approach is now feasible. However, the lengths of oligothat can be synthesized using the column-based approach is limited to upto only 200 nucleotides (Kosuri, Nature Methods, 11(5): 499:507(2014)).Other high-throughput oligo synthesis approaches have proliferatedrecently. Microarray-based approaches that also utilize phosphoramiditesynthons are attractive for large scale synthesis of shortoligonucleotide strands (Science, 251: pp. 767-773 (1991); Proc. Natl.Acad. Sci., 91: pp. 5022-5026 (1994)). Photolithographic techniques areleveraged in array-based oligo synthesis approaches to selectivelydeprotect phosphoramidite precursors. Ink-jet based printing ofnucleotides on microarray surfaces greatly increases the throughput ofoligo synthesis (Nature Biotechnology, 19: 342:347 (2001)).

Template-free synthesis of DNA was discovered very early inbiochemistry, noted by Arthur Kornberg. Other early examples includetemplate free RNA polymerization with Qbeta replicase. Terminaldeoxynucleotidyl transferase (TdT; terminal transferase) and telomeraseare two more examples in biology where deoxynucleic acid (DNA) synthesiscan occur in the absence of a DNA template, meaning that no first strandis needed (see, for example, U.S. Pat. Nos. 8,808,989 and 8,071,755, andU.S. Publication Nos. 2009/0186771, and 2011/0081647, and 2013/0189743).In the case of TdT, synthesis occurs in a 5′ to 3′ direction from aninitiator primer and appends on deoxyribonucleic acid triphosphates(dNTPs) available in the surrounding solution. The TdT releases from thetemplate after one or a few incorporations, and will a new polymerasewill come on to continue affixing new nucleotides. Currently, sequencecontrol of the incorporation of the nucleotides is achieved by additionof a single nucleotide to a solution, washing, and adding the nextnucleotide in a cycle of additions of homopolymers.

Single-stranded Binding protein (SSB) is a protein found in many livingsystems and can bind non-specifically to single-stranded DNA. It iscommercially available from New England Biolabs (NEB). For example, NEBoffers highly thermostable ssDNA binding proteins that are ideal fornucleic acid amplification and sequencing (Tth RecA, NEB #M2402; and ETSSB, NEB #M2401). NEB also offers ssDNA proteins for use invisualization of DNA structures with electron microscopy and screeningof DNA libraries (E. coli RecA, NEB #M0249, NEB #M0355) and to improverestriction enzyme digestion and enhance the yield of PCR products (T4Gene 32 Protein, NEB #M0300).

Peptide synthesis on insoluble solid-support, pioneered by Robert BruceMerrifield (J. Am. Chem. Soc., 85(14): pp. 2149:2154 (1963)), is thestandard method to synthesize peptides. A free N-terminal amine iscoupled to an N-protected amino acid unit. The protecting group is thencleaved to introduce a free amino group to which another N-protectedamino acid can be linked. The peptide is grown on the solid-support thenfinally cleaved to obtain the free synthesized peptide. Optional washingsteps can be added for each step in the cycle to remove excess reagentsfrom the column. The lengths of peptides that can be synthesized usingthe column approach is limited to 30-70 amino acid residues. Longerpolypeptides are realized by using native chemical ligation to “stitch”two or more polypeptides together.

Biotin is a small chemical adduct that can attached covalently to DNA atthe 5′ or 3′ end or added covalently to proteins. Streptavidin is aprotein that binds biotin tightly with ˜10-14 mol/L Kd and this systemis often used to attach proteins or DNA to a solid phase composed of asurface or to beads that can be manipulated through physicalinteractions, such as magnetically active beads. Many other methods ofcovalent or non-covalent attachment to solid-phase or surface supportsare known in the art. Enzymes can also be controlled using temperatureand small molecules including divalent ions such as magnesium, or drugmolecules to either inhibit, decelerate, accelerate, or otherwisecontrol their activity in vitro for functional applications such asprogrammed synthesis. Standard restriction enzymes also offer a way ofmanipulating synthesized DNA, for example, to cleave and release anucleic acid from a substrate, etc., and in a sequence-specific mannerwhen practical.

Microfluidics technologies exist for automated control of fluid movementactuated by various means. For example, Electrowetting On Dielectric(EWOD) is a method to control the movement of single picoliter tonanoliter droplets controlled through motive force by induced electricpotential at the sight of the move (Sensors and Actuators A: Physical,95(2-3), pp. 259-268 (2002)). Typically, a droplet of aqueous solutionis held at a location by an induced electric potential on a dielectric.This droplet can be moved by moving the potential to a second adjacentlocation. By applying equal potential, the droplet can be split ormerged, and movement of the droplet can induce mixing. Alternatively,the droplets in the EWOD device are steered by optical excitation of theelectrode which creates a potential that induces droplet motion. Theoptical source can be shaped to create potential gradients to actuatethe droplets in different directions. However, current methods usingEWOD are restricted by the area of the EWOD surface, and the volume ofthe drop.

Digital information storage as sequences of nucleic acids is of interestin the storage market for archival memory storage Church, et al.,Science; V. 337, (6102), pp. 1628 (2012); Goldman, et al., Nature, v.494, pp 77-80 (2013); Zhirnov, et al., Nature Materials, V.15, pp366-370 (2016)). Methods of extraction of specific memory from a poolhave also previously been implemented (Yazdi, et al., Scientific ReportsV.5, Article number: 14138 (2015); Bornholt, et al., IEEE Micro 37 (3);pp. 98-104 (2017); and Organick, et al., Nature Biotechnology, V36, pp.242-248 (2018)), specifically showing the use of polymerase chainreaction with a hash table set of barcodes to amplify specific sequencesfrom a pool. This approach is limited by the pool size that can be useddue to PCR cross reactivity and amplification of spurious sequences thatdistract from the targeted sequence. Further, each data selection usinga PCR-based approach either requires the extraction of the aliquot fromthe original sample, ultimately having to resynthesize the entiresample, or contaminates the original sample by introduction of enzymes.

There is a need for methods of biopolymer synthesis that are moreefficient, more automatable, produce longer biopolymer strands, orcombinations of these features.

There is also a need for methods of automated encapsulation ofbiopolymers for scalable, separable archival storage.

There is also a need for methods of barcode synthesis to retrieve theencapsulated product that can be dynamically allocated and rewrittenwithout modifying the encapsulated product (such as a protectedbiopolymer).

Therefore, it is an object of the invention to provide systems andmethods for automated synthesis of user-defined sequence-controlledbiopolymers.

It is also an object of the invention to provide methods to dynamicallyalter biopolymer sequences using cutting enzymes or chemically-specificphoto-degradation, followed by de novo enzymatic synthesis.

It is also an object of the invention to provide methods tosimultaneously produce multiple distinctly addressed sequence-controlledbiopolymers having distinct sequences and sizes.

It is also an object of the invention to provide fully automated systemsand methods for large-scale synthesis of addressed biopolymers havinguser-defined sequence and size.

It is a further object of the invention to provide uniquely addressedsynthesized biopolymers of user-defined sequence and size.

It is an object of the invention to provide methods of encapsulation ofsequence-controlled biopolymers.

It is also an object of the invention to provide fully or partiallyautomated systems and methods for pooling sequence-controlledbiopolymers and encapsulating the pool into an encapsulated block.

It is also an object of the invention to provide fully or partiallyautomated systems and methods for barcoding encapsulated blocks,removing the barcode, and/or re-attaching a barcode of the same ordifferent sequence in a repeated way.

It is also an object of the invention to provide methods for selectivemodification of biopolymers.

It is also an object of the invention to provide fully or partiallyautomated methods for the generation of barcode nucleic acid sequencesof defined and adjustable melting temperatures.

It is also an object of the invention to provide methods of usingfluorescent probe sequences complementary to barcode sequences toidentify encapsulated blocks using fluorescence or other opticalsignature.

It is also an object of the invention to provide methods of usingfluorescent probe sequences to sort encapsulated blocks.

It is a further object of the invention to provide methods ofdynamically barcoding encapsulated blocks for retrieval and computation.

SUMMARY OF THE INVENTION

Methods for the scalable, automated, template-free synthesis, and/ormodification of biopolymers using microfluidics systems have beendeveloped. The methods optionally include encapsulation and dynamicmolecular barcoding of nucleic acids and other biopolymers having aprogrammed sequence and size. Methods of using the synthesizedbiopolymers for archival storage, retrieval, modification, organizationand re-organization of encoded data through movement of fluids using amicrofluidic system are also provided.

The methods utilize microfluidic liquid handling technology fortemplate-free synthesis and manipulation of biopolymers such as nucleicacids. In some forms, the methods enable massively parallelized nucleicacid synthesis with each location on a microfluidic platform growing anindependent, geometrically addressed, long single-stranded nucleic acidby programmed movement of droplets containing nucleotides that aresequentially incorporated into the 3′ end of the growing nucleic acid.The methods achieve the droplet cycling needed in theaddition/de-protection steps for enzymatic DNA, RNA, and peptidesynthesis. The methods optionally incorporate magnetic and/ortemperature control globally or locally on the microfluidic platform, toenable additional control over the synthesis. Analogous methods canproduce and/or modify sequences of numerous types of biopolymers usingdifferent component building blocks (such as monomers).

Exemplary microfluidic and liquid handling systems that can be employedfor the methods include Electrowetting on Dielectric (EWOD) devices,acoustic droplet distribution devices, volumetric displacementdistribution devices, ink-jet type fluidic distributors, or any otherdevice that actuates micro-fluidic flow across a chip, for example,using microwells or synthetic compartments. A preferred microfluidicdevice is an EWOD chip.

In some forms, the methods generate biopolymers of programmed sequenceand length in the absence of a template sequence. An exemplarybiopolymer is single-stranded nucleic acid of greater than 200nucleotides in length, for example, 500 nucleotides, 1,000 nucleotides,or 10,000 nucleotides, or greater than 10,000 nucleotides, for exampleup to 100,000 nucleotides in length. The methods optionally include thesteps of purifying, amplifying, encapsulating, sequencing,functionalizing, and/or otherwise manipulating the synthesizedbiopolymers. In some forms, the methods add, remove, or modify one ormore molecular sequence tags or barcodes within a biopolymer. In someforms the methods add, remove or modify one or more molecular sequencetags or barcodes on an encapsulated biopolymer. Some or all of themethod steps can be carried out using a computer-controlled EWOD chip.

Typically, the methods for synthesizing biopolymers include the steps of(a) combining on a microfluidic device a droplet including a componentinitiation sequence with one or more droplets collectively comprising acomponent building block and an attachment catalyst to form a combineddroplet; and (b) repeating step (a) to perform the step-wise addition ofcomponent building blocks to the biopolymer to form a biopolymer havinga preselected, desired biopolymer sequence and length.

In an exemplary method, synthesis is carried out using movement ofdroplets actuated buy an Electrowetting on Dielectric (EWOD)microfluidic chip. Generally, the droplets including a componentinitiation sequence and each of the droplets collectively including thecomponent building block and the attachment catalyst are, prior to thecombining, at different locations on the EWOD chip. Generally, one ormore additional droplets, each including an additional componentbuilding block, are at different locations on the EWOD chip than thedroplet including the component initiation sequence, the dropletscollectively including the component building block and the attachmentcatalyst, or the combined droplet. Generally, the combining includesconditions suitable for the attachment catalyst to attach the componentinitiation sequence to the component building block to form abiopolymer.

In some forms, the methods include the steps of (a) selecting a desiredbiopolymer sequence; (b) providing the component building blocks,attachment catalyst, component initiation sequence, wash reagents, andstop reagents as discrete droplets on a microfluidic device; (c)identifying the route and conditions for each droplet to combine withthe other droplets to perform the step-wise addition, removal, ormodification of building blocks to form a polymer having the desiredbiopolymer sequence; and (d) performing the step-wise addition, removal,or modification of building blocks to form a polymer having the desiredbiopolymer sequence according to the route identified in (c).

In some forms the methods optionally include the steps of isolating thebiopolymer having the desired sequence from the microfluidic device.Exemplary attachment catalyst/agents include polymerase enzymesincluding TdT, Q-beta replicase, and teleomerase.

In some forms, the methods include the step of forming one or more ofthe droplets containing the component initiation sequence and thedroplets collectively including the component building block and theattachment catalyst by splitting the droplets from reservoirs thatcollectively include the component initiation sequence, the componentbuilding block, and the attachment catalyst. In some forms, the methodsinclude the step of forming one or more of the additional droplets bysplitting the additional droplets from reservoirs that collectivelycomprise the additional component building blocks.

Methods of modifying a pre-existing biopolymer are also provided. Forexample, in some forms the methods attach component building blocks to abiopolymer to add one or more sections to one or more regions of thebiopolymer. In other forms, the methods remove component building blocksfrom a biopolymer, for example, to remove one or more sections from thebiopolymer. In some forms, the methods attach or remove a section to abiopolymer that is a molecular barcode. One or more molecular barcodescan be synthesized or attached to one or more positions of a biopolymer.

In some forms, the methods include one or more steps to alter thechemical or structural properties of synthesized single-stranded nucleicacid sequences. Therefore, methods for functionalizing single-strandednucleic acid sequences using microfluidic systems are also provided. Insome forms, methods include steps of functionalizing a newly-synthesizedbiopolymer by one or more processes that alter chemical or structuralproperties of the biopolymer. In some forms, chemical or structuralproperties of a newly-synthesized single-stranded nucleic acid aremodified, for example, through addition of one or more oligonucleotideaddress sequences. In an exemplary form, methods of functionalizingsingle-stranded nucleic acids include conjugating a functionalizednucleic acid to the newly-synthesized nucleic acid prior to releasing orpurifying the nucleic acid from the EWOD device.

In some forms, the methods manipulate a biopolymer to dynamicallyremove, modify, and/or attach one or more components. In some forms, themethods manipulate a section of a biopolymer that functions as amolecular barcode. For example, in some forms, the methods degrade abarcode site-specifically using cutting enzymes, or targetedphoto-degradation, or other targeted cleavage, followed by elongatingthe polymer de novo to generate a new barcode sequence.

In some forms the methods include one or more steps to encapsulate abiopolymer. Encapsulation can be carried out using a material suitablefor the encapsulation of the biopolymer. Preferably the encapsulationprocess occurs following polymer synthesis, and prior to purification.In some embodiments, two or more biopolymers are encapsulated together.Therefore, the step of encapsulating biopolymer(s) can include one ormore steps of organizing, sorting and selecting biopolymers forencapsulation. In some forms, two or more biopolymers are encapsulatedtogether according to identification of a common feature. An exemplarycommon feature is one or more components (e.g., sequences) that arecommon to molecular barcodes in two or more biopolymers.

In some forms optical activation of nucleotide precursors containingoptically-cleavable functional groups that are known in the art is usedto control nucleotide precursors incorporated by the enzyme (Mathews, etal., Org Biomol Chem. 14(35), pp. 8278-88 (2016)). In some forms, themethods modify nucleotides or other biopolymer subunits to improve theincorporation of additional moieties, or to facilitate sequencing. Forexample, in some forms, the methods include addition of hydrophobicmoieties or conductive moieties to a biopolymer.

In some forms, the methods include substrates immobilized onto a solidsupport or surface. For example, in some forms, the methods include oneor more component initiation sequences, a catalyst enzyme, and/or abiopolymer immobilized onto a solid support. In some forms, when asolid-support system is used, the methods employ continuous flow systemsto actuate movement of substrates. For example, the growing biopolymercan be isolated from the continuous flow in a droplet that is containedwithin a covering material, for example, formed by a lipid or otherchemical matrix. Access to the droplet including the immobilizedinitiator sequence, the catalyst enzyme, or biopolymer is controlled,for example, by opening or closing channels through the cover materialor by direct penetration through the cover material.

In some forms, the methods include the step of encapsulating abiopolymer within an encapsulating agent. In other forms, the methodsinclude the step of degrading or otherwise removing an existingencapsulating agent from one or more regions of the biopolymer. Forexample, in some forms, the methods remove an encapsulating agent, thenremove, add, or substitute one or more sequences or other components ofthe biopolymer, then re-encapsulate the modified biopolymer in the sameof different encapsulating agent.

In some forms, the step of purifying the synthesized nucleic acids fromthe microfluidic device includes polymerase chain reaction (PCR). Forexample, PCR using the desired sequence as a scaffold can be used toamplify and/or purify the desired sequence from the EWOD chip. In someforms, the length of the scaffold is 100 or more nucleotides in length,e.g., 1,000 nucleotides in length; 1,500 nucleotides in length; 2,000nucleotides in length; 2,500 nucleotides in length; 3,281 nucleotides inlength; 10,000 nucleotides in length; 12,000 nucleotides in length; orgreater than 12,000 nucleotides.

In some forms, the biopolymer is functionalized by introduction offunctionalized component building blocks into the solution. Exemplaryfunctional components include fluorescent moieties, radio-labeledmoieties, and magnetic moieties. In an exemplary form, modifiednucleotides are used as component building blocks for nucleic acidpolymer synthesis. Exemplary modified nucleotides include Cy5fluorophore-modified nucleotides, phosphorothioate-modified nucleotides,and deoxyuridines.

Methods of using EWOD-based template-free synthesis for the parallel,simultaneous synthesis of multiple different biopolymers are provided.For example, in some forms, individual biopolymers having apre-programmed length and sequence are prepared at individual locationson the same EWOD chip to simultaneously produce multiple independent,geometrically addressed, biopolymers. In an exemplary method, longsingle-stranded DNA is synthesized by programmed movement of dropletscontaining the nucleotide that will next be incorporated into the 3′location. This technology is broadly applicable to the same dropletcycling needed in the addition/deprotection steps of chemical DNA, RNA,and peptide synthesis. Incorporation of magnetic and/or temperaturecontrol globally or locally on the dielectric chip offers additionalutility for control over the synthesis. Compositions of biopolymerssynthesized according to the described methods are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an EWOD device that shows the reagentreservoirs and channel addressing of the reagents for parallelized DNAsynthesis. For illustrative purposes, the channels are drawn to show thepath of the droplets. In other forms, the channels are removedcompletely and the droplets are created and moved by an optical source.The channels can contain, but not limited to, the enzyme, the nucleotideprecursors, the reaction initiator, a capping reagent, a washingreagent, and a chemical to halt enzymatic activity. The channels areattached to a collection reservoir where the DNA is capture forsubsequent use.

FIG. 2 is a schematic of movement of the droplets from necessary tosynthesize a DNA fragment of sequence ATCG. This sequence of moves canbe generalized to any nucleic acid sequence incorporation. It is shownwith 4 wells containing dATP (“A”), dTTP (“T”), dCTP (“C”), and dGTP(“G”), 2 buffer wells, a release solution well, a collector output port,and a waste port. A magnetic bead with streptavidin bound to abiotinylated initiator strand is at B-3. Each of A, T, C, and G alsocontain buffer, salt, and template free polymerase (e.g., TdT). The gridlayout and series of instructions to build the polymer “ATCG” are shown.In addition to the generality of the sequence that can be built, this isparallelizable across the EWOD chip, allowing for simultaneous growth ofdifferent sequences in as many addresses would be available per chipsize.

In some forms, the methods synthesize and/or manipulate nucleic acidbarcodes. For example, in some forms, the methods implement a scheme formolecular identification that includes mutations in the barcode forsimilar terms. In some forms, multiple point mutations within a nucleicacid sequence that is a barcode are combined to provide a moleculardatabase of barcode. Therefore, in some forms, blocks ofsequence-controlled biopolymers can be addressed by differentidentifying barcodes that are themselves separate sequence-controlledbiopolymers that represent the metadata encoded by a memory object,similar to a “molecular hash”. In some forms, the methods introduce setsof point mutations in barcodes. Therefore, in some forms the methodsenable more similar polymer-blocks to be extracted from the solutionmore readily than sequences that are not similar. For example in oneexemplary form, a 25-mer barcode sequence is selected to berepresentative of “red” and a separate 25-mer barcode sequence isselected to be representative of “blue” (exemplary barcodes aredescribed in the article entitled “Design of 240,000 orthogonal 25merDNA barcode probes”, by Xu, et al., Proc Natl Acad Sci, 106 (7)2289-2294 (2009)). Point mutations are made to make the barcode lesssimilar to the original barcode, and reverse complements of each areobtained. A melting temperature is determined (e.g., by quantitativePCR) for each primer pair corresponding to metadata of “red”s,“like-red”s, “blue”s, and “like-blue”s, respectively. High meltingtemperatures indicate perfect complementarity, while the nearbyneighbors indicate selections could include non-specific (i.e., “fuzzy”,or “noisy”) retrieval of corresponding metadata.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “nucleotide” refers to a molecule that contains a base moiety,a sugar moiety and a phosphate moiety. Nucleotides are typically linkedtogether through their phosphate moieties and sugar moieties creating aninter-nucleoside linkage. The base moiety of a nucleotide can beadenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), andthymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or adeoxyribose. The phosphate moiety of a nucleotide is pentavalentphosphate. A non-limiting example of a nucleotide would be 3′-AMP(3′-adenosine monophosphate) or 5′-GMP (5′-guanosine monophosphate).

The term “residue” of a chemical species refers to the moiety that isthe resulting product of the chemical species in a particular reactionscheme or subsequent formulation or chemical product, regardless ofwhether the moiety is actually obtained from the chemical species. Thus,an ethylene glycol residue in a polymer refers to one or more —OCH₂CH₂O—units in the polymer, regardless of whether ethylene glycol was used toprepare the polyester. As another example, in a polymer of monomersubunits, the incorporated monomer subunits can be referred to asresidues of the un-polymerized monomer.

The term “nucleotide analog” refers to a nucleotide which contains sometype of modification to the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties. There are many varieties of these typesof molecules available in the art and available herein.

The term “nucleotide substitute” refers to a nucleotide molecule havingsimilar functional properties to nucleotides, but which does not containa phosphate moiety. An exemplary nucleotide substitute is peptidenucleic acid (PNA). Nucleotide substitutes are molecules that willrecognize nucleic acids in a Watson-Crick or Hoogsteen manner, but whichare linked together through a moiety other than a phosphate moiety.Nucleotide substitutes are able to conform to a double helix typestructure when interacting with the appropriate target nucleic acid. Itis also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, interactionwith DNA. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Exemplary conjugates include but are not limited tolipid moieties such as a cholesterol moiety.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areinterchangeable and refer to a deoxyribonucleotide or ribonucleotidebiopolymer, in linear or circular conformation, and in either single- ordouble-stranded form. For the purposes of the present disclosure, theseterms are not to be construed as limiting with respect to the length ofa biopolymer. The terms can encompass known analogues of naturalnucleotides, as well as nucleotides that are modified in the base, sugarand/or phosphate moieties (e.g., phosphorothioate backbones, lockednucleic acid). In general and unless otherwise specified, an analogue ofa particular nucleotide has the same base-pairing specificity; i.e., ananalogue of A will base-pair with T. When double-stranded DNA isdescribed, the DNA can be described according to the conformationadopted by the helical DNA, as either A-DNA, B-DNA, or Z-DNA. The B-DNAdescribed by James Watson and Francis Crick is believed to predominatein cells, and extends about 34 Å per 10 bp of sequence; A-DNA extendsabout 23 Å per 10 bp of sequence, and Z-DNA extends about 38 Å per 10 bpof sequence.

In some cases nucleotide sequences are provided using characterrepresentations recommended by the International Union of Pure andApplied Chemistry (IUPAC) or a subset thereof. IUPAC nucleotide codesinclude, A=Adenine; C=Cytosine; G=Guanine; T=Thymin; U=Uracil; R=A or G;Y=C or T; S=G or C; W=A or T; K=G or T; M=A or C; B=C or G or T; D=A orG or T; H=A or C or T; V=A or C or G; N=any base; “.” or “-”=gap. Insome forms the set of characters is (A, C, G, T, U) for adenosine,cytidine, guanosine, thymidine, and uridine respectively. In some formsthe set of characters is (A, C, G, T, U, I, X, Ψ) for adenosine,cytidine, guanosine, thymidine, uridine, inosine, uridine, xanthosine,pseudouridine, respectively. In some forms the set of characters is (A,C, G, T, U, I, X, Ψ, R, Y, N) for adenosine, cytidine, guanosine,thymidine, uridine, inosine, uridine, xanthosine, pseudouridine,unspecified purine, unspecified pyrimidine, and unspecified nucleotide,respectively.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

The terms “cleavage” and “cleaving” of nucleic acids, refer to thebreakage of the covalent backbone of a nucleic acid molecule. Cleavagecan be initiated by a variety of methods including, but not limited to,enzymatic or chemical hydrolysis of a phosphodiester bond. Bothsingle-stranded cleavage and double-stranded cleavage are possible, anddouble-stranded cleavage can occur as a result of two distinctsingle-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered “sticky” ends. In certainforms cleavage refers to the double-stranded cleavage between nucleicacids within a double-stranded DNA or RNA chain.

Nucleotide and/or amino acid sequence identity percent (%) is understoodas the percentage of nucleotide or amino acid residues that areidentical with nucleotide or amino acid residues in a candidate sequencein comparison to a reference sequence when the two sequences arealigned. To determine percent identity, sequences are aligned and ifnecessary, gaps are introduced to achieve the maximum percent sequenceidentity. Sequence alignment procedures to determine percent identityare well known to those of skill in the art. Often publicly availablecomputer software such as BLAST, BLAST2, ALIGN2 or MEGALIGN (DNASTAR)software is used to align sequences. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyformulas needed to achieve maximal alignment over the full-length of thesequences being compared. When sequences are aligned, the percentsequence identity of a given sequence A to, with, or against a givensequence B (which can alternatively be phrased as a given sequence Athat has or comprises a certain percent sequence identity to, with, oragainst a given sequence B) can be calculated as: percent sequenceidentity=X/Y100, where X is the number of residues scored as identicalmatches by the sequence alignment program's or formula's alignment of Aand B and Y is the total number of residues in B. If the length ofsequence A is not equal to the length of sequence B, the percentsequence identity of A to B will not equal the percent sequence identityof B to A. Mismatches can be similarly defined as differences betweenthe natural binding partners of nucleotides. The number, position andtype of mismatches can be calculated and used for identification orranking purposes.

The term “endonuclease” refers to any wild-type or variant enzymecapable of catalyzing the hydrolysis (cleavage) of bonds between nucleicacids within a DNA or RNA molecule, preferably a DNA molecule.Non-limiting examples of endonucleases include type II restrictionendonucleases such as Fold, HhaI, HindIII, NotI, BbvCl, EcoRI, BglII,and AlwI. Endonucleases comprise also rare-cutting endonucleases whenhaving typically a polynucleotide recognition site of about 12-45basepairs (bp) in length, more preferably of 14-45 bp. Rare-cuttingendonucleases induce DNA double-strand breaks (DSBs) at a defined locus.Rare-cutting endonucleases can for example be a homing endonuclease, amega-nuclease, a chimeric Zinc-Finger nuclease (ZFN) or TAL effectornuclease (TALEN) resulting from the fusion of engineered zinc-fingerdomains or TAL effector domain, respectively, with the catalytic domainof a restriction enzyme such as Fold, other nuclease or a chemicalendonuclease including CRISPR/Cas9 or other variant and guide RNA.

The term “exonuclease” refers to any wild type or variant enzyme capableof removing nucleic acids from the terminus of a DNA or RNA molecule,preferably a DNA molecule. Non-limiting examples of exonucleases includeexonuclease I, exonuclease II, exonuclease III, exonuclease IV,exonuclease V, exonuclease VI, exonuclease VII, exonuclease VII, Xm1,and Rat1. In some forms, an enzyme is capable of functioning both as anendonuclease and as an exonuclease. The term “nuclease” generallyencompasses both endonucleases and exonucleases, however in some formsthe terms “nuclease” and “endonuclease” are used interchangeably hereinto refer to endonucleases, i.e., to refer to enzyme that catalyze bondcleavage within a DNA or RNA molecule.

The term “ligating” refers to enzymatic reactions in which twodouble-stranded DNA molecules are covalently joined, for example, ascatalyzed by a ligase enzyme.

The terms “aligning” and “alignment” refer to the comparison of two ormore nucleotide sequence based on the presence of short or longstretches of identical or similar nucleotides. Several methods foralignment of nucleotide sequences are known in the art, as will befurther explained below.

The term “nucleic acid capture” refers to binding of any nucleic acidmolecule of interest having complementary nucleic acid sequences to acorresponding sequence associated with a separate nucleic acid, orhaving affinity for the sequence employed, and being immobilized orattached to a solid support matrix. For example, “RNA capture” refers tobinding of any ribonucleic acid molecule of interest to thecomplementary sequence on a nucleic acid coupled to a solid supportmatrix.

The phrase that a molecule “specifically binds” to a target refers to abinding reaction which is determinative of the presence of the moleculein the presence of a heterogeneous population of other biologics. Thus,under designated immunoassay conditions, a specified molecule bindspreferentially to a particular target and does not bind in a significantamount to other biologics present in the sample. Specific binding of anantibody to a target under such conditions requires the antibody beselected for its specificity to the target. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity. The term “specificbinding”, for example, between two entities, means an affinity of atleast 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M-1. Affinities greater than 10⁸ M-1are preferred.

The term “targeting molecule” refers to a substance which can direct asynthesized biopolymer to a receptor site on a selected cell or tissuetype, can serve as an attachment molecule, or serve to couple or attachanother molecule. The term “direct” refers to causing a molecule topreferentially attach to a selected cell or tissue type. This can beused to direct cellular materials, molecules, or drugs, as discussedbelow.

The terms “antibody” and “immunoglobulin” include intact antibodies, andbinding fragments thereof. Typically, fragments compete with the intactantibody from which they were derived for specific binding to an antigenfragment, including separate heavy chains, light chains Fab, Fab′F(ab′)2, Fabc, and Fv. Fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes a bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, Clin.Exp. Immunol., 79:315-321 (1990); Kostelny, et al., J. Immunol., 148,1547-1553 (1992).

The terms “epitope” and “antigenic determinant” refer to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10, amino acids, in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance.

The term “small molecule,” as used herein, generally refers to anorganic molecule that is less than about 2,000 g/mol in molecularweight, less than about 1,500 g/mol, less than about 1,000 g/mol, lessthan about 800 g/mol, or less than about 500 g/mol. Small molecules arenon-polymeric and/or non-oligomeric.

The term “droplet” refers to a distinct volume of a fluid that isdistinct and separate from, and independently movable from, otherdroplets. Fluid droplets are generally formed by splitting a volume offluid from a reservoir containing a larger volume of the same fluid.

The terms “attachment reagent,” “attachment catalyst/agent,” “assemblyreagent,” “catalyst,” “assembly catalyst,” “attachment catalyst,” and“catalyst reagent” refer to a reagent that actuates, enhances,increases, or otherwise enables the addition of a component buildingblock onto an initiator sequence or onto a growing biopolymer.Typically, the attachment of a component building block by a catalyst iscontrolled by movement of one or more fluid droplets according to anEWOD device. An exemplary molecule that specifically enhances theaddition of one or more nucleotide building blocks to a growing nucleicacid biopolymer is a template-free polymerase. Exemplary attachmentagents include TdT, Qbeta replicase, and telomerase enzymes.

The terms “building block” and “component building block” refer to adiscrete component of the biopolymer that is formed by step-wiseaddition to an initiator. Building blocks are typically basic structuralunits of biopolymers, such that biopolymers result from the step-wiseassembly of the building blocks. Exemplary building blocks includenucleotides, amino acids, monosaccharides and polypeptides. In someforms, building blocks are monomers. In other forms, building blocks aremultimers, such as dimers, homodimers, heterodimers, oligomers etc.Exemplary multimers of basic structural units include short nucleic acidsequences, di-peptides, tri-peptides, and oligosaccharides.

The terms “initiator,” “initiator sequence,” “component initiationsequence,” and “initiating oligomer” refer to a discrete sequence ofcomponent building blocks that acts as an initiation molecule for thestep-wise template-free assembly of component building blocks forsynthesis of a user-defined biopolymer. In some forms, the initiatormolecule includes one or more recognition sequences for an attachmentcatalyst. An exemplary initiator sequence is an oligonucleotideincluding a nucleic acid sequence that is a recognition sequence of aTdT enzyme.

The term “sequence,” in the context of the disclosed biopolymers, refersto the order of building blocks, such as nucleotides, in the biopolymer.For example, common DNA has a sequence of nucleotide building blockschosen from A, C, G, and T. Biopolymers made from other types ofbuilding blocks will have sequences defined by the order of thosebuilding blocks in the biopolymer.

The term “bead” or “magnetic bead” refers to a solid structure that isused as a support matrix for one or more reagents when used in methodsfor synthesis of biopolymers. Beads can be any suitable bead.

The terms “wash reagent,” “wash buffer,” “wash,” and “rinse solution”refer to a solution that is used to purify remove one or more reagentsfrom a biopolymer, initiator or catalyst. Typically, the wash buffer isa solvent that is effective to solvate and remove reagents from amolecule that is immobilized, for example, an immobilized biopolymer.The wash buffer can be contacted with a droplet of solution, or can bethe solvent used to dissolve one or more reagents, for example, toreduce or prevent the activity of the reagent.

The term “wash conditions” refers to the environmental/externalconditions under which combination with a wash reagent (i.e., a distinct“wash step”) is carried out. For example, a wash can be carried out bycombining one or more wash reagents with a solution or immobilizedsupport containing the biopolymer or initiator, and subsequent exposureof the combined solution to one or more environmental/externalconditions. Exemplary conditions include the time of combination, theamount and concentration of each wash reagent, exposure to agitation,exposure to heat, light, vapor, changes in pressure, changes inelectrical charge, etc.

The term “stop reagents” refers to a reagent that selectively ornon-selectively reduces or prevents the activity of an active agent. Forexample, a stop-reagent can have a pH or contain a molecule thatinterferes with the activity of an enzyme. Typically, stop reagentschange the parameters of a solution into which they are mixed, forexample, to change pH, change temperature, change ion concentration,competitively bind to an active site on an active agent, etc. In someforms, stop reagents selectively bind and/or sequester co-factorsnecessary for enzyme function. Exemplary stop reagents include acids,bases, ionic solutions and glycerol. In some forms, stop reagentsimmediately prevent or impede one or more attachment reactions, forexample, by inhibiting the activity of the catalyst enzyme, or bysequestering or otherwise reducing/altering the concentration ofcomponent building blocks available for addition.

The term “stop conditions” refers to the environmental/externalconditions under which combination with a stope reagent (i.e., adistinct “stop step”) is carried out. For example, stop conditions caninclude combining one or more stop reagents with a solution orimmobilized support containing the biopolymer or initiator, andsubsequent exposure of the combined solution to one or moreenvironmental/external conditions. Exemplary conditions include the timeof combination, the amount and concentration of each wash reagent,exposure to agitation, exposure to heat, light, vapor, changes inpressure, changes in electrical charge, etc.

The term “blocking reagents” refers to a reagent that specificallyblocks a chemical reaction, for example, to prevent the addition of anamino acid to a growing poly-peptide biopolymer. Typically, blockingreagents add a chemical “cap,” or other molecule to the terminalcomponent building block in the biopolymer “chain”. The cap selectivelyprevents the addition of a subsequent component building block at therespective location on the biopolymer. The term “unblocking reagents”refers to any agent that reverses, reduces, or otherwise abrogates theeffects of a blocking reagent. Unblocking agents are typically not washreagents. Rather, unblocking agents actively modify the biopolymer toenable, induce or enhance the attachment of a component building blockat a site that was previously blocked.

The term “attachment conditions” refers to the conditions under whichthe user-defined attachment of component building blocks to aninitiator, or to the terminal component building block of a biopolymer(i.e., a distinct “attachment step”) is carried out. For example,attachment can be carried out by combining the attachment agent with theinitiator or biopolymer and one or more component building blocks underconditions amenable to the function of the catalyst. Exemplaryconditions include the time of combination, the amount and concentrationof each reagent, ionic concentration, presence of any necessaryco-factors, absence of stop reagents, exposure to agitation, exposure toheat, light, vapor, changes in pressure, changes in electrical charge,etc.

The terms “encapsulating”, “enveloping”, “coating”, “covering”, and“shelling” are used interchangeably to refer to the process by whichbiopolymers, and optionally additional agents, are completely orpartially enclosed by an encapsulating agent. The term “encapsulatingagent” refers to a molecular entity, such as a polymer or other matrix.

The terms “microfluidic device”, “microfluidics”, “microfluidic chip”,and “microfluidic platform” refer to any device, or system that supportsand/or enables or actuates the movement of sub-microliter volumes offluids, for example, as discrete droplets. Typically, microfluidicdevices implement components and means for controlling the user-definedsplitting, movement, and combining of discrete fluid droplets in acontrolled manner, as well as modifying or altering one or morephysicochemical properties, such as temperature, electric charge, light,magnetic force, etc. In some forms, microfluidic devices control themovement, behavior and manipulation of fluids through one or more meansfor actuating fluid movement. Exemplary microfluidic devices actuatefluid movements through mechanisms including continuous flow, fluiddispensing, EWOD, pressure, optical or combinations thereof.Microfluidic devices can be “open” (i.e., fluid is contained, moved andmanipulated on a single surface), or “closed” (i.e., fluid is contained,moved and manipulated between two surfaces). In some forms, the term“microfluidic device” is used interchangeably with “microfluidicsystem”, and includes the means for inputting user-defined control offluid manipulation (e.g., through a general-user interface that employscomputer software to control the movement of fluids within the device).The term “microfluidic system” also refers to additional equipment, suchas equipment that is external to apparatus for controlling fluidmovement, for example, devices for controlling parameters such astemperature, light, pressure, humidity, etc. In some form, “microfluidicdevices” include devices and systems to input data for control of themovement or manipulation of the droplets on a microfluidic platformlocated close to, or at a distance from the site of data input. In someforms, the data input device is or incorporates a computer. In someforms, the system or device includes one or more systems for providinginformation to the control system, e.g., a device for proving feedback.In some forms, data input is autonomous (e.g., computational tasks canbe performed, autonomously, like programs that run on conventionalsilicon computers, but here in the liquid state).

The terms “EWOD”, or “Electrowetting” refers to the technique ofElectrowetting on dielectric (EWOD) to control the movement of singlepicoliter to nanoliter droplets, e.g., through motive force by inducedelectric potential at the sight of the move (Sensors and Actuators A:Physical, 95(2-3), pp. 259-268 (2002)). The terms “EWOD chip”, “EWODplatform”, or “EWOD device” refer to a platform or similar equipment,for actuating the movement of fluids by the EWOD phenomenon. Anexemplary EWOD chip is a microfluidic chip, such as a digitalmicrofluidic chip. EWOD chips can be “open” (i.e., fluid droplets moveacross a surface without a layer above the fluid), or “closed” (i.e.,fluid droplets move across a surface with a second layer above thefluid).

II. Methods for Automated Synthesis and Manipulation of Biopolymers

Systems and methods for the automated, step-wise synthesis and/ormanipulation of a biopolymer having a user-defined sequence/structureand size have been established. The systems and methods do not require apre-existing template sequence or structure. The methods generallyinvolve step-wise assembly of distinct component building blocks (e.g.,nucleotides, amino acids, monosaccharides, etc.) onto a componentinitiation sequence as droplets at one or more discrete locations on amicrofluidic platform. In some forms, the methods synthesize and/ormanipulation of user-defined sequences of nucleic acids (e.g., DNA orRNA) using a grid-addressable location in a sequence-specified manner inan absence of a template on an electrowetting-on-dielectic (EWOD) chip.

The addressed position of the growing polymer strand is determined bythe position on a microfluidic platform, such as an EWOD chip. In someforms the growing biopolymer is held stationary on the microfluidicplatform by fixing a component initiation sequence to a surface at theaddressed location, or fixing the component initiation sequence to amagnetic bead and holding it in location by a strong magnet. Theoperating temperature can be varied according to the requirement of thesynthesis. User-defined movement of droplets (e.g., through the electricpotential induced by an EWOD chip) droplets containing componentbuilding blocks, buffers, and attachment catalyst, are moved andcombined and mixed with the droplet containing the growing biopolymersequence chain.

An exemplary catalyst is a template-free polymerase enzyme for theassembly of a nucleic acid. Upon combining appropriate droplets, theenzyme attaches available nucleotides to the 3′ end of the polymer (see,for example, Biochimica et Biophysica Acta, 1804(5): pp. 1151-1166(2010)). Droplets including one or more component building blocks arecombined with the enzyme solution and are sequentially incorporated ontothe growing biopolymer chain. Either by limiting the nucleic acid numberavailable per reaction, or by removing the nucleotides and solution byremoving the droplet but keeping the sequence fixed in its addressedgrid location and washing 1, 2, 3, or more than 3 times with dropletscontaining just water or just buffer and salts will allow for programmedtime stops of reactions.

Because microfluidic platforms, such as EWOD chips, are typically smallin grid size, and can be simultaneously moved and controlled bypreprogramming the steps of merging, mixing, and separating, abiopolymer having a pre-defined programmed sequence can be grown at theaddressed locations. The movement, splitting, and merging of droplets isnot limited to electrical operation (e.g., as implemented through anEWOD device), but can also be actuated utilizing optical control toperform operations using droplets. Thus, by increasing the size of thechip to include more grid points, 1 strand, 1,000 strands, 1,000,000strands or more can be synthesized simultaneously. Because the TdTenzyme is only limited by occlusion from the 3′ end by thesingle-stranded DNA, the growing polymer can be of size 100 nts, 1,000nts, up to 10,000 nucleotides, or more than 10,000 nts.

In preferred forms, the assembly process is mediated by the activity ofone or more attachment catalysts. Therefore, control of the assemblyprocess is mediated by the rate and activity of the attachment catalyst.Attachment catalysts are selected according to the nature of thebiopolymer that is the desired end-product of the synthesis. Exemplaryattachment catalysts include enzymes (e.g., polymerases, phosphatases,esterases, lipases, glycosyl-transferases, and proteases), acids, aswell as external conditions such as light (e.g., photo-switchedassembly), air and heat. In other forms, the assembly process occurs inthe absence of an attachment catalyst. For example, if the componentbuilding blocks are polypeptides, proteins, nanostructures, etc.,assembly can occur through interaction specific or non-specificinteraction between the initiator element and the component buildingblock. An exemplary non-catalyzed assembly is the dimerization followinginteraction between two G actin proteins.

In some forms, the methods synthesize polymers onto one or more solidsupport matrices. In some forms, the component initiation sequence iscoupled to a magnetic bead to facilitate the step-wise assembly process.The solid support anchors the initiator sequence in a user-determinedaddress location on the microfluidic device, enabling the step-wisemovement of reagents onto and away from the initiator sequence asrequired to achieve optimal assembly. When the component initiationsequence is coupled to a solid support, methods for assembling thebiopolymer can include iterations of microfluidic device-mediatedmovement of aqueous droplets to sequentially combine the componentinitiation sequence with droplets containing different reagents.Therefore, in some forms the step of combining the initiator sequenceand one or more component building blocks includes sequentialcombination of the immobilized initiator sequence with one or moredroplets including one or more reagents including wash buffers,component building blocks, assembly catalysts, buffers, blockingreagents, and/or stopping reagents. Each microfluidic device-mediatedcombination and separation event can be repeated one or more times toselectively combine/mix or separate/exclude one reagent from another.For example, the step-wise assembly of each building block can becarried out as a cycle including microfluidic device-mediated movementof droplets to combine an subsequently separate the immobilizedinitiator sequence with (1) wash buffer; (2) a component building blockand assembly catalyst and optionally one or more buffers required forthe assembly catalyst to combine the component building block with theinitiator sequence; (3) a blocking reagent and/or stopping reagent toprevent the activity of the assembly catalyst, and (4) a wash buffer.The cycle can be repeated to sequentially add each component buildingblock to the growing biopolymer. Factors such as the timing between eachmicrofluidic device-mediated movement of droplets, and externalconditions can be optimized according to the requirements of eachbiopolymer. The biopolymer remains attached to the solid support matrixthroughout the cyclic assembly process, and can be cleaved away from thesupport matrix following addition of the last component building block.

In some forms, a software program is used to coordinate the microfluidicdevice-mediated movement of droplets.

Typically, the methods include one or more of the following steps:

(a) Selecting a target polymer;

(b) providing reagents as droplets on a microfluidic device, thereagents including

-   -   (i) a component initiation sequence;    -   (ii) one or more component building block(s); and    -   (iii) an attachment catalyst;

wherein the a component initiation sequence is provided as a separatedroplet from the component building blocks;

(c) combining a droplet comprising the component initiation sequencewith one or more droplet(s) comprising a component building block and anattachment catalyst to form a combined droplet,

wherein the combining comprises conditions suitable for the attachmentcatalyst to attach the component initiation sequence to the componentbuilding block to form a biopolymer.

In some forms the methods further include the steps of

-   -   (i) Blocking or otherwise reducing, stopping or preventing the        attachment of the component building block to the biopolymer;    -   (ii) Washing the biopolymer with one or more wash buffers to        reduce or remove one or more reagents from the developing or        completed biopolymer; and    -   (iii) Modifying the biopolymer, for example, by addition or        removal of one or more functional motifs.

In some forms the methods further include the steps of

(d) Purifying or otherwise isolating the biopolymer from the EWOD chip.

(e) Confirming or assessing the microfluidic device-synthesizedbiopolymer. Confirming the biopolymer can include sequencing oramplifying the completed biopolymer.

A. Selecting a Target Biopolymer

The methods synthesize a sequence-controlled “target” biopolymer havinguser-defined sequence and size using addressed locations on anmicrofluidic device. Methods for microfluidic device-based template-freesynthesis of target biopolymers from corresponding component buildingblocks provide the ability to simultaneously synthesize multiplebiopolymers having the same or different sequences using the samemicrofluidic device. Automated synthesis can be carried out for one ormore biopolymers simultaneously on the same microfluidic device frominstructions input as a sequence of droplet movements corresponding touniquely addressed locations on the chip.

The step of selecting a target biopolymer generally includes the stepsof: (1) determining the number and composition of biopolymers to besynthesized; (2) rendering a microfluidic platform as a grid network;and (3) assigning a unique address to each node identified byintersecting grid-lines on the network. In some forms, biopolymers aresynthesized at a single location on the microfluidic device grid.Biopolymers can be addressed according to the node/location of synthesison the grid network. Therefore, in some forms, the methods include thestep of assigning a unique address to each biopolymer.

1. Selecting Number and Composition of Biopolymers

Methods for the programmable microfluidic device-mediated template-freesynthesis of a user-defined biopolymer require the user-defined input ofthe sequence and size of the desired biopolymer. In some forms,biopolymer sequences are selected based upon one or more designcriteria. In other forms biopolymer sequences are selected randomly.

The step-wise assembly of component building blocks onto an initiatorsequence is aided when the relative location of each component buildingblock is determined in one or more distinct fluid reservoirs on themicrofluidic device to enable the appropriate coordinated movement ofdroplets. Therefore, in some forms the methods require input parametersthat define the target sequence(s) to be synthesized. Input can be inthe form of a computer-readable program. Therefore, in some forms, thestarting point for the synthesis process is the identification of thetarget sequence. When multiple polymers having the same or differentsequences are required, the user must designate each sequence as havinga specific location on the microfluidic device for the synthesis tooriginate.

In an exemplary form, the user-defined sequence is a nucleic acid, andthe reservoirs of component building blocks that are addressed areselected according to the number of different nucleotide bases to beincorporated into the biopolymer. For example, synthesis of a DNAsequence would typically require at least four distinct reservoirs ofcomponent building blocks, one for each of the main nucleobases found inDNA (i.e., one reservoir for each of adenine, cytosine, thymine, andguanine), as well as one or more reservoirs for each of the appropriateassembly catalyst (i.e., a template-free polymerase enzyme), a reactionbuffer, one or more wash buffers (e.g., water), as well as a stoppingbuffer (e.g., to deactivate the polymerase enzyme). Some reagents usedin the methods can be combined in the same reservoir or kept in separatereservoirs. Some reagents, such as individual nucleotides to be added inparticular sequence order, should be in separate reservoirs from eachother.

The number of different biopolymers that is to by synthesized is alsoconsidered. The methods enable the automated synthesis of up to1,000,000 different polymers on the microfluidic device. In an exemplaryform, the methods synthesize ten different nucleic acids, each includingup to four different nucleobases, and having a different size/length.Each of the different polymers is assigned a uniquely addressedreservoir (e.g., each reservoir is assigned a number between 1 and 10,inclusive, each integer corresponding to a single initiator sequence)and each of the reagents is assigned a unique integer (e.g., 1-4 foreach nucleobase, 5-7 for polymerase enzyme and each of two buffers, 8-9for each of two wash buffers, and 10 for a stop buffer, respectively).Accordingly, in the exemplary method, at least 20 nodes are required asdistinct reagent reservoirs on the microfluidic device.

Methods for loading reagents to a specific location or reservoir in amicrofluidic device, e.g., an EWOD chip, are known in the art, and theskilled person will understand the loading protocol can vary accordingto the type and size of microfluidic device that is employed, as well asthe force through which droplet isolation and movement are actuated.

a. Conversion of Data to Biopolymer Sequence

In some forms, the methods include providing a biopolymer sequence thatencodes a piece of desired information, such as bitstream data. Anexemplary sequence-controlled polymer encoding information as bitstreamdata is a nucleic acid, such as single or double-stranded DNA, or RNA.For example, in some forms, a single-stranded nucleic acid sequenceencoding user-defined bitstream data is input for the design of anucleic acid. In some forms, a portion or portions of a digital formatof information, such as an html format of information or any otherdigital format such as a book with text and/or images, audio, or moviedata, is converted to bits, i.e., zeros and ones. In some forms, theinformation can be otherwise converted from one format (e.g., text) toother formats such as through compression by Lempel-Ziz-Markov chainalgorithm (LZMA) or other methods of compression, or through encryptionsuch as by Advanced Encryption Standard (AES) or other methods ofencryption. Other formats of information that can be converted to bitsare known to those of skill in the art.

Schemes and systems for encoding data in the form of a sequence, such asa biopolymer, are known in the art. Therefore, the described methods caninclude the step of converting data into or encrypting data within thesequence of one or more biopolymers. For example, in some forms, thestep of inputting data includes steps of converting data into abiopolymer sequence. The corresponding sequence is subsequently used asinput to coordinate the movement of droplets required for synthesis ofthe biopolymer.

2. Rendering a Microfluidic Device as a Grid Network

The methods require data input to coordinate the appropriate movement ofdroplets on a microfluidic device that can actuate movement ofsub-microliter volumes of fluid as independent droplets to mediatepolymer synthesis. In preferred forms, the microfluidic device is adevice for actuating movement of sub-microliter droplets via EWOD. Anexemplary EWOD device is an EWOD chip. The initial step in the processincludes an assembly process, whereby the chip is rendered as a networkgrid, representing the relative locations of the channels and reservoirson the chip.

To coordinate the step-wise assembly process, the chip is rendered as anetwork grid, representing the relative locations of the channels andreservoirs on the chip. For example, each vertex (node) of the networkis represented by a point of intersecting/overlapping grid lines(interacting edges). For example, each vertex (node) of the network isassigned an address based on the intersection of corresponding gridlines. Each node represents the potential position, or destination of adroplet. Each line, or “edge” represents the potential passage of adroplet when it moves between the nodes connected by that edge. Anexemplary grid network for a microfluidic device chip is represented inFIG. 1. The schematic in FIG. 1 depicts each channel for fluid movementas an edge on the grid. Each node is addressed according to its relativelocation. Generally, a fraction of the total number of nodes on a chipare addressed as reservoirs for reagents. The schematic grid representedin FIG. 1 depicts the outermost nodes as reservoirs for reagents. Insome forms, the address of each node is determined and automaticallyassigned from input parameters, for example, a total number of channelson each side of the microfluidic device. Exemplary addressing schemesfor each vertex include alpha-numeric (e.g., a, b, c and 1, 2, 3, etc.).

The number of nodes available for droplet interface on the microfluidicdevice is proportional to the number of channels (“edges” in a node-edgenetwork defined by the grid graph of the chip). A grid network having anodes on one axis and b nodes on another axis (a×b grid graph) has thevertex set [a]×[b], and edges of two types: horizontal edges(i,j),(i+1,j) (of which there are (a−1)b); and vertical edges(i,j),(i,j+1) (of which there are a(b−1)), for a total of ab verticesand (a−1)b+a(b−1)=2ab−a−b edges. In some forms, each node is assigned asingle integer value, for example, each node in a 10×10 grid is assigneda number from 1 to 100, inclusive. In some forms, each node is assigneda dual integer address, for example, each node in a 10×10 grid isassigned an address such as (a, 1) or (j, 10), etc.

3. Assigning Unique Addresses to Nodes at Intersecting Grid Lines in theNetwork

In some forms, the channels that define edges of the grid network on thechip are physical channels (e.g., groves or recesses between reservoirswithin the microfluidic device). In other forms, the channels are“virtual” channels, for example, where movement of droplets between thenodes of the grid is actuated by optical force.

Employing virtual channels for optical movement of droplets on themicrofluidic device grid surface can greatly increase the number ofaddressed nodes that can be represented on a microfluidic device havingdefined dimensions, as compared with the potential maximum number ofphysical channels on a microfluidic device of equal dimensions.Therefore, in some forms, the separation and movement of droplets on themicrofluidic device actuated by optical movement of droplets increasesthe number of “channels” and nodes on the grid relative to the number ofnodes and channels on a microfluidic device (e.g., an EWOD chip) ofequal size where the droplets are actuated by physical force. Therefore,in some forms, the methods assign a grid network having between 4 and10,000,000 nodes, inclusive, to a microfluidic device. The number ofnodes on the grid network correlates to the number of addressed nodes onthe microfluidic device. The number of addressed nodes on themicrofluidic device is directly proportional to the number ofbiopolymers that can be simultaneously synthesized on the microfluidicdevice. Therefore, in some forms, the methods include providing theaddresses of up to 1,000,000 nodes at independent locations at on thesame microfluidic device, for example, between 1 and 10 nodes, between 1and 100 nodes, between 1 and 1,000 nodes, between 100 and 10,000 nodes,between 1,000 and 100,000 nodes.

When a node on the microfluidic device is the location of a reagentreservoir, the address of the node is used as input to direct theautomated splitting and movement of droplets containing reagents fromthe corresponding reservoir. Therefore, the address of a node can beassociated with one or more reagents. In some forms, when a nodecontains one or more immobilized component initiation sequence(s), theaddress of the node is the address of the corresponding synthesizedbiopolymer. In some forms, the step of assigning discrete addresses foreach location on the grid network of the microfluidic device.

B. Providing Reagents as Discrete Fluid Droplets

The methods require utilizing microfluidic splitting and movement offluid droplets containing reagents as solutions on a microfluidic device(e.g., actuated by EWOD on an EWOD chip). Therefore, the methods requireproviding reservoirs of substrates at addressed locations on amicrofluidic device.

In some forms, growing biopolymer is immobilized at an addressedlocation on the microfluidic device. For example, in some forms, thecomponent initiation sequence or the catalyst includes one or moresequences designed to hybridize or otherwise bind to stationary-phaseobjects such as magnetic beads, surfaces, agarose or other polymerbeads. In other instances, the component initiation sequence or thecatalyst includes one or more sites for conjugation to a molecule. Forexample, the component initiation sequence or the catalyst can beconjugated to a protein, or non-protein molecule, for example, to enableaffinity-binding of the component initiation sequence or the catalyst,or of the synthesized polymer.

1. Providing Addressed Reagents

The methods include providing reagents as droplets split from largerfluid reservoirs on a microfluidic device. The size, concentration andposition of fluid reservoirs is varied according to the reagent, thesynthesis protocol, and the dimensions of the microfluidic device.

a. Providing Fluid Reservoirs

The methods include control of reagents as droplets split from largerfluid reservoirs on a microfluidic device. Each fluid reservoir on themicrofluidic device can contain one or more reagents. Reservoirs aretypically addressed according to the grid of the microfluidic device,and the relative location (address) of the reservoir forms part of theinput data used to control and direct the microfluidic device-basedsynthesis. Parameters of droplets such as the fluid volume andconcentration of reagents within each reservoir can be selectedaccording to the specific requirements of the synthesis that is desired.Typically, the volume and concentration of a reagent reservoir used formicrofluidic device-mediated fluid movement is proportional to thenumber, volume and concentration of droplets that are required to besplit from the reservoir for synthesis to be completed. An exemplaryfluid reservoir volume is between 1 nanoliter (1 nl) and 100 milliliters(100 ml), for example, between about 1 microliter (1 μl) and about 100microliters (100 μl). A typical synthesis will have 10 μl reservoircontaining, for example, 8 μM concentration of each monomer buildingblock in a different reservoir and a reservoir containing 100 μl ofbuffer, and other 10 μl reservoirs containing 1 μM initiator sequences,and other 10 μl reservoirs containing 10 μM template-free polymerase,such as TdT.

b. Providing Fluid Droplets

The methods include movement and combination of reagents as droplets.Parameters of droplets such as volume and concentration can be selectedaccording to the specific requirements of the synthesis that is desired.Typically, the volume of a droplet used for microfluidic device-mediatedfluid movement is between about 0.1 Picoliter (pl), and about 100microliters (μl), for example, between about 1 pl and about 50nanoliters (nl). In an exemplary form, each droplet size is betweenabout 0.5 NL and five NL. The concentration of reagents within eachdroplet is between about 0.1 femtomolar (1 fM) and about 100 micromolar(100 μM). In an exemplary form, the droplets contain reagents formicrofluidic device-based synthesis of user-defined addressed nucleicacids. The amount of initiator sequence nucleic acid in a droplet isbetween about 1 femtomol (1 fmole; 10⁻⁵ moles) and 1,000 picomoles(1,000 pmoles; 10⁻⁹ moles) per 1 picoliter (1 pL) droplet size, up to 5nanoliter (5 nL) droplet size, and beyond. A typical synthesis will havedroplets either of 50 pL or 1 nL with concentrations of the initiatorderived from the reservoir or diluted out of the reservoir,approximately 10 μM for the polymerase, 1 μM for the initiator, and 8 μMfor the nucleotides, as one example.

C. Combining Droplets to Coordinate Biopolymer Synthesis

The methods include identifying the sequence of movement for reagentsnecessary to achieve fluid-based template-free synthesis of biopolymers.Typically, the movement enables the splitting, relocation andcombination of droplets to achieve the step-wise assembly of the entirebiopolymer sequence, based on the address information provided in thecorresponding grid network. Therefore, the methods provide routinginformation for each of the droplets to complete the step-wise assemblyof each biopolymer.

Any system that provides control of the coordinated movement of discretesub-microliter amounts of fluids can be used to synthesis biopolymeraccording to the described methods. Exemplary systems are microfluidicsystems and devices. Exemplary systems that can be employed for thedistribution and movement of small fluid volumes as independent dropletsaccording to the described methods include EWOD devices, acousticdroplet distribution devices, such as the commercially available Echo555, volumetric displacement distribution devices, such as the Mosquitopipette robot, or ink-jet type fluidic distributors. Additionally, thesynthesis may occur by flow across a chip, with microwells or syntheticcompartments used for synthesis. In a preferred form, microfluidicdevices/systems that employ electrowetting on dielectric (EWOD) actuatedmovement of sub-microliter fluid droplets are used for synthesis ofbiopolymers according to the described methods.

Methods for optical fluid motion are known in the art. In some forms,the methods employ fluid motion that results from the dynamic thermalexpansion in a gradient of viscosity. For example, the viscosity of afluid at a given spot is reduced by its enhanced temperature. This leadsto a broken symmetry between thermal expansion and thermal contractionin the front and the wake of the spot. As result the fluid movesopposite to the spot direction due to both the asymmetric thermalexpansion in the spot front and the asymmetric thermal contraction inits wake.

1. Electrowetting on Dielectric (EWOD) Techniques

In some forms, the assembly of biopolymers through step-wise addition ofuser-defined building block components occurs through EWOD-mediatedmovement of droplets containing substrates, enzymes, wash buffers andother reagents. The extent and direction of the movement of each dropletcoordinates the combination of two or more droplets at any givenlocation on the EWOD chip. The methods render an EWOD chip as a grid,with each discrete location at the intersection of one or more of thegrid lines as a distinctly addressed location on the chip. Therefore,movement of droplets from one discrete addressed location on the EWODchip to another discrete addressed location on the chip can be carriedout as a computer-readable program to synthesize biopolymers having aprogrammable user-defined composition.

Electrowetting describes the electromechanical reduction of a liquid'scontact angle as it sits on an electrically-charged solid surface. Whenan electric field is applied across the interface between a solid and awater droplet, the surface tension of the interface is changed,resulting in a change in the droplet's contact angle. In oil ambient(i.e., when the water droplet is surrounded by oil rather than air), theelectrowetting effect can provide >100° of reversible contact anglechange with fast velocities (>10 cm/s) and low electrical energy (˜100to 102 mJ/m² per switch).

Electrowetting has become one of the most widely used tools formanipulating tiny amounts of fluids on surfaces. A large number ofapplications based on electrowetting have now been demonstrated,including lab-on-a-chip devices, optics, and displays.

An important parameter in electrowetting studies is Young's angle (θY),defined as follows:

cos θY=(γod−γad)/γao  (1)

where; γod is the interfacial tension between the electrowetting liquid(a, typically aqueous) and the oil (o) surrounding the electrowettedliquid; γad is the interfacial tension between (a) and the dielectriclayer (d); and γao is the interfacial tension between (a) and (o).

For most electrowetting applications, it is generally desirable to uselow voltages (V) to switch from Young's angle to the electrowettedcontact angle (θV). Low-voltage operation is particularly important forparticular displays, such as e-paper displays, that require very largearrays (thousands or millions) of electrodes. These devices requireactive-matrix electrode control. Active matrix control makes use of thinfilm transistors (TFTs) that independently address each of the pixelstates. TFTs typically provide reliable operation up to about only 15V.However, achieving reliable electrowetting devices operating at ≤15V hasbeen a considerable challenge.

In an electrowetting system, Young's angle is reduced to theelectrowetted contact angle (0V) as predicted by the electrowettingequation,

cos θV=(γod−γad)/γao+εV2/(2dγao)  (2)

where: ε is the dielectric constant and d is the thickness of thedielectric; γ is used for terms denoting the interfacial tension betweenthe electrowetting liquid, the oil, and the dielectric, as described inequation 1, above; and V is the applied DC or AC RMS voltage.

Once surface tensions are optimized for a high Young's angle (θY), theelectrowetting equation predicts that lower voltages may be obtainedonly by reducing the thickness of the dielectric, or by using adielectric with a higher dielectric constant. A change in contact angleon the order of 100 degrees is desirable for good electrowetting devicefunction.

The methods require control of movement of reagents as droplets splitfrom larger fluid reservoirs on an EWOD chip. Mechanisms for controllingextent and direction of movement of droplets using EWOD technology areknown in the art. Exemplary mechanisms for actuating movement ofdroplets include electrical charge and optical control systems.

a. Optical Electrowetting Techniques

In some forms, movement of droplets on EWOD is actuated by an opticalforce. By optically modulating the number of carriers in thespace-charge region of the semiconductor, the contact angle of a liquiddroplet can be altered in a continuous way. This effect can be explainedby a modification of the Young-Lippmann equation. Exemplary methods foroptical movement of droplets include optoelectrowetting, andphoto-electrowetting. Optical (light-manipulated) EWOD technology offersfull programmability of droplet movement at the single-droplet level forup to millions of droplets simultaneously and instantaneously. Anexemplary technology is the, OPTOSELECT™ technology, that useslow-intensity visible light to precisely manipulate cells, beads andreagents, commercially available from Berkeley Lights. OPTOSELECT™consumable chips contain thousands of nanoliter pens, allowing theannotation and characterization of individual droplets.

i. Opto-Electrowetting

Optoelectrowetting (OEW) involves the use of a photoconductor. Wheretraditional electrowetting runs into challenges, however, such as in thesimultaneous manipulation of multiple droplets, OEW presents a lucrativealternative that is both simpler and cheaper to produce. OEW surfacesare easy to fabricate, since they require no lithography, and havereal-time, reconfigurable, large-scale manipulation control, due to itsreaction to light intensity.

By shining an optical beam on one edge of a liquid droplet, the reducedcontact angle creates a pressure difference throughout the droplet, andpushes the droplet's center of mass towards the illuminated side.Control of the optical beam results in control of the droplet'smovement.

Using 4 mW laser beams, OEW has proven to move droplets of deionizedwater at speeds of 7 mm/s. Traditional electrowetting requires atwo-dimensional array of electrodes for droplet actuation. The largenumber of electrodes leads to complexity for both control and packagingof these chips, especially for droplet sizes of smaller scales. Whilethis problem can be solved through integration of electronic decoders,the cost of the chip would significantly increase

ii. Photo-Electrowetting

Photoelectrowetting (PEW) uses a photo capacitance and can be observedif the conductor in the liquid/insulator/conductor stack used forelectrowetting is replaced by a semiconductor.

Photoelectrowetting using the photo capacitance in aliquid-insulator-semiconductor junction is achieved via opticalmodulation of carriers in the space charge region at theinsulator-semiconductor junction that acts as a photodiode—similar to acharge-coupled device based on a metal-oxide-semiconductor. Droplettransport is achieved by focusing a laser at the leading edge of thedroplet. Droplet speeds of more than 10 mm/s can be achieved without thenecessity of underlying patterned electrodes.

In some forms methods for synthesis of biopolymers on EWOD employphotoactivated electrowetting-actuated movement of droplets. Typically,the methods employ a hydrophobic surface to enable movement of sessiledroplets. An exemplary system for PEW includes a photoactive wafer thatcan be photoactivated to induce an electric field covered with adielectric which actuates the droplet.

b. EWOD Synthesis on Solid Support

In some forms, a growing biopolymer is immobilized at an addressedlocation on the EWOD chip, such that movement of the biopolymer is notmediated by EWOD. For example, in some forms, the component initiationsequence or the catalyst includes one or more sequences designed tohybridize or otherwise bind to solid support or stationary-phase objectssuch as magnetic beads, surfaces, agarose or other polymer beads. Inother instances, the component initiation sequence or the catalystincludes one or more sites for conjugation to a molecule. For example,the component initiation sequence or the catalyst can be conjugated to aprotein, or non-protein molecule, for example, to enableaffinity-binding of the component initiation sequence or the catalyst,or of the synthesized polymer.

When a solid support or stationary-phase object is used, the mechanismfor moving droplets is distinct from, and does not induce movement ofthe solid support or stationary-phase object, such that droplets can bemoved onto, or split from the immobilized reagent(s).

2. Providing Input for Microfluidic-Based Synthesis

In some forms the methods include inputting instructions for themovement of droplets on the pre-defined network grid of the microfluidicdevice to assemble each user-defined polymer using a computer-basedinterface. For example, in some forms, data corresponding to theaddressed nodes of the network are input to a computer for the automatedsynthesis of one or more biopolymers on the microfluidic device.

Methods for inputting coordinates of a grid network in computer-readableform are known in the art. For example, in some forms the methodsinclude providing the geometric parameters that define the grid networkon the microfluidic device and/or the address of each reservoir of areagent required for the synthesis of each biopolymer. Geometricparameters include the spatial coordinates of all vertices, the edgeconnectivity between vertices, and the faces to which vertices belong.

The extent of automation of control of microfluidic device-mediatedmovement of droplets can be varied from complete automation (e.g.,random selection of target sequence and size, based on pre-determinedgrid coordinates for a microfluidic device having pre-addressedreservoirs having standard volumes of each reagent), to no automation(each step of droplet splitting and node to node movement of droplets isuser-defined for a user-defined grid custom designed to includeuser-supplied reagents). In some forms, the input data includes only theaddress of each immobilized component initiation sequence (i.e., thelocation at which each biopolymer will be synthesized), and the desiredtarget sequence. Input data controlling movement of droplets to achievethe cycle of adding each component building block (e.g., coordinatedwashing, adding component building blocks, catalysts, blockingcatalysts), the number of cycles required, etc. is pre-programed, orotherwise provided independently. In other forms, input data controllingeach node-to-node movement of a droplet throughout the entire synthesisprocess is also input, for each biopolymer.

Following sequence design, grid-determination and input of theinstructions necessary for the microfluidic device-based synthesis ofbiopolymers according to the described methods, the addressed biopolymersequences are synthesized, optionally functionalized and purified on themicrofluidic device. Therefore, methods for the microfluidicdevice-based template-free synthesis of biopolymers having user-definedsequence include the step of producing the biopolymers. In some forms,the methods simultaneously synthesize up to 1,000,000 biopolymers atindependently addressed locations on the same microfluidic device, forexample, between 1 and 10 polymers, between 1 and 100 polymers, between1 and 1,000 polymers, between 100 and 10,000 polymers, between 1,000 and100,000 polymers.

Typically, parameters are determined as input data for each synthesis.Exemplary parameters include (a) the sequence of movement of droplets tocontact the initiator sequence with each reagent in the appropriateorder for synthesis of the desired biopolymer sequence, as well as (b)the conditions required for optimal activity of the reagent at each stepof the synthesis.

Typically, the methods attach component building blocks to an initiatorto synthesize a biopolymer having a user-defined sequence of componentbuilding blocks. Because the number of component building blocks that isattached to growing biopolymer cannot be controlled at the level of eachindividual molecule, the resulting biopolymers produced by each completesynthesis will typically include a bell curve for the number ofcomponent building blocks attached to the biopolymer molecules duringeach cycle. For example, in some forms, each attachment reaction mayattach between zero and one hundred component building blocks to theinitiator or biopolymer. Typically, the average number of componentbuilding blocks attached at each stage is one or two. In someexperiments, the average number of component building blocks attached ateach stage is eight and follows a Poisson distribution around 8additions. Typically the number of homopolymer additions is controlledby the amount of precursors available and the ratio between the growingpolymer and the available nucleotides, and the temperature of operation,and the buffer used, and the enzyme used. In some forms, thedistribution of the number of building blocks attached at each stage iscontrolled, for example, by limiting the factors that enhance theattachment process. Exemplary factors that can be controlled include theconcentration of substrates, catalysts, ions, and other reagents, aswell as incubation times, and variation of other factors includinglight, agitation, temperature, pressure, electrical charge, etc.

In some embodiments, the time of each reaction step is determined bysimulating the Michaelis-Menten equation for estimating the nucleotideusage. In further embodiments, the estimation of the number of additionsneeded to differentiate one sequence controlled polymer from another isdetermined by simulating the number of additions assuming a Poissondistribution.

In some embodiments, the addition of the nucleotide is blocked byoptically activatable nucleotide analogs. In one implementation, thenucleotides or addressed strands will become activated to allow for thenext incorporation by the specific projection of light, such as from aDLP chip (Texas Instruments). In some implementations, the specificnucleotide or polymer will be activatable based on the wavelength of thelight used, such that some polymers or nucleotides become active onlywhen, for example, blue light is used.

a. Sequences and Cycles of Droplet Movement

The assembly is carried out by step-wise movement of fluid droplet on asuitable microfluidic device surface. In preferred forms, the movementof droplets is carried out using a EWOD device. Movement of droplets onan EWOD device can be actuated by application of electric charge, or byoptical force. Movement includes splitting of droplets from largervolumes, for example, to provide discrete volumes of reagents that aremixed in the appropriate quantities in an appropriate reaction volume tocontrol attachment and biopolymer synthesis. In preferred forms, thereagents are split and combined in an amount effective to maximize theyield and correct assembly of the biopolymer.

The examples of DNA polymer synthesis can generally be applied to DNA orRNA synthesis using alternative enzymes such as Telomerase or Qbetareplicase. Additionally the examples herein describe droplet-basedmovement using EWOD, but are generally applicable to droplet merging,separating, and mixing offered by other devices such as through opticalcontrol, for example using fluid moved by a laser-scanning microscope.

In some forms, the methods initiate and complete synthesis of abiopolymer by step-wise addition of reagents to an initiator sequencethat is maintained at a single location on a microfluidic device. Inother forms, initiation and completion of the synthesis of a biopolymerby step-wise addition of reagents to an initiator sequence includesmicrofluidic device-based movement of a droplet containing the initiatorsequence and growing biopolymer. Synthesis can be carried out in aqueoussolution without a solid support or matrix, or can include one or morereagents immobilized onto a solid support or matrix.

In other forms, a growing biopolymer is immobilized at an addressedlocation on the microfluidic device. For example, in some forms, thecomponent initiation sequence or the catalyst includes one or moresequences designed to hybridize or otherwise bind to solid support orstationary-phase objects such as magnetic beads, surfaces, agarose orother polymer beads. In other instances, the component initiationsequence or the catalyst includes one or more sites for conjugation to amolecule. For example, the component initiation sequence or the catalystcan be conjugated to a protein, or non-protein molecule, for example, toenable affinity-binding of the component initiation sequence or thecatalyst, or of the synthesized polymer.

When a solid support or stationary-phase object is used, the mechanismfor moving droplets is distinct from, and does not induce movement ofthe solid support or stationary-phase object, such that droplets can bemoved onto, or split from the immobilized reagent(s).

In an exemplary form, a sequence of microfluidic device-mediatedsplitting, movement and combination of droplets enables assembly of anucleic acid from fluid reservoirs containing an enzyme catalyst,component building blocks (e.g., nucleotides), and a componentinitiation sequence (e.g., oligonucleotide), respectively. In a firstmovement, droplets are simultaneously split from the enzyme (E+I), andone or more nucleotide (N1T, N2T, etc.) reservoirs. In a secondmovement, the droplets are merged to form a combined droplet. Thecombined droplet is incubated for 1 minute to achieve the reactionforming a product (“N1”). In a third movement, the droplet containing N1is moved to the next droplet containing the next nucleotide reagent. Themovement of droplets to split, steer, and merge fluids can be actuatedby electrical potential (e.g., as in an EWOD device), or by opticalexcitation.

Typically, input parameters include instructions for the electrical oroptical actuated initiation (splitting of a droplet from a reservoir),and directional of node-node movement of a droplet. The input parametersalso include the amount of time between subsequent movement or splittingevents at any given node (address on the grid). Therefore, parameterssuch as incubation time, amount of reagent added or removed, and thetotal volume of droplets at each location can be controlled, eitherdirectly, or as a pre-programed template of instructions for eachmicrofluidic device.

i. Solid Support-based Synthesis

In some forms, the methods synthesize biopolymers from multipleconsecutive cycles of step-wise assembly of the component buildingblocks from an initiator sequence that is coupled to a solid support.The solid support can be a particle, such as a bead, that is loaded ontoor otherwise present on the microfluidic device, or it can be a surfaceof the microfluidic device. The initiator sequence can be coupled to thesolid support using any bond, material, or system known in the art forconjugating molecules together. In a preferred form, the initiatorsequence is coupled to a solid support using the biotin/streptavidinconjugation system, for example, via a biotin sequence at the 5′ regionof the initiator tag (i.e., 5′-biotinylated initiator sequence).

An exemplary sequence of movement includes the steps of (1) combining acomponent building block with an initiator sequence; (2) combining anattachment reagent with the droplet containing a component buildingblock with an initiator sequence to form an attachment reaction droplet;(3) optionally combining a buffer with the attachment reaction dropletto initiate, enhance or otherwise control the attachment; (4) combininga stop reagent with the attachment reaction droplet to stop theattachment; (5) optionally combining a wash reagent with the reactiondroplet to create a washed reaction droplet; (6) splitting the majorityof the washed reaction droplet to create a waste droplet and a washedbiopolymer droplet; and repeating step (5) one or more times tothoroughly wash the biopolymer. Generally, the cycle including each ofsteps (1)-(6), above, is repeated for the addition of each componentbuilding block to the developing biopolymer.

Therefore, in some forms, the number of cycles required to construct thebiopolymer is equal to the size of the sequence that is synthesized.

Each of the movement steps (1)-(6), above, can be further characterizedby the sequence of (i) splitting of a droplet containing the fluid fromthe corresponding reservoir; (ii) moving the droplet to the location ofa target droplet; and (iii) combining the droplet with the targetdroplet. In some forms, the target droplet contains the biopolymer, orthe initiator. In other forms, the target droplet does not contain thebiopolymer or the initiator. Therefore, in some forms each movement stepcan involve multiple steps of splitting, moving, and combining, forexample, to prepare a droplet having a desired composition prior tocombining with the biopolymer or the initiator.

One or more of the catalyst enzyme and/or initiator sequence can beimmobilized or attached to one or more solid support matrices. In someforms, the addressed synthesis is carried out on a passivated surface orslide, for example, a slide that has the initiator and polymer on asurface, or in a picoliter-scale well etched into a slide. In someforms, the initiator sequence or the attachment enzyme is attached to asurface or a well by, for example, biotin, or other methods known in theart. In some forms, the initiator sequence and enzyme will be accessibleto a lateral flow of washing solution or component building blocks(e.g., nucleotides). In such cases, the addressed growing strand will beprogrammed for the next incorporation by focused light on the surfaceusing, for example, a 4 k DLP chip.

In some embodiments, the synthesis of the polymer will occur within awell or micrometer scale vesicle separated from an outside environmentby the presence of a lipid bilayer or polymer mesh. In such embodiments,the mesh or layer can allow or disallow the crossing of building blocksby an external motive force, such as by electroporation orelectrophoresis. This again can be addressed by circuit based design,creating the potential needed to allow for crossing the barrier to entryinto the encapsulated region. In such cases, the encapsulated regionwould be 1-10 micrometers, and be similar to synthetic cells. In suchcases, the growing polymer may be DNA or proteins or RNA and mayencoding for genetic or information elements.

ii. Continuous Flow-Based Synthesis

Attaching the polymerase or catalyst or component initiation sequence tothe surface of a chip by passivating the chip using techniques known inthe art additionally allows continuous flow incorporation of componentbuilding blocks (e.g., nucleotides) to the growing polymer. In someforms, the initiator sequence and enzymes are segregated in differentwells having micro-meter or nano-meter dimensions, with singlepolymerases and initiators within the well. Flow of the individualmonomers can be controlled or diverted using electronic switches,heating, or through lithographic plates, or through coverage with lipidbilayer with or without embedded protein channels. Access to thewell/solution containing the enzyme is controlled in order to directsynthesis of the biopolymer. Exemplary methods to control access to thewell/solution containing the enzyme include direct penetration throughthe membrane or cover of the well, or by activating one or more channelsthrough the cover or membrane.

In some forms, combining single or multiple component building blockswith the well/solution containing the enzyme is accomplished throughactivating a potential, for example, by using electric potential acrossthe membrane to allow for the flowing nucleotides to pass through thesurface (similar to electroporation that is well known, but in a micro-or nano-scale well) or by inducing an electric signal to activate aprotein channel, or an electric potential that causes nucleotide ornegatively charged monomers, or positively charged monomers to passinside of an otherwise closed surface, such as electroporating throughagarose, acrylamide, or other polymers. Therefore, in some forms, thewell contains the initiator or growing polymer and polymerase thatcannot pass out of the well due to blockade from a bilayer or chemicalmesh. In some forms one or more of the channels may be opticallycontrolled for nucleotide or polymer layer crossing using opticalpatterning.

iii. Solution-Based Synthesis

In some forms, the growing polymer is not affixed to beads or a surface,but is free in solution. For example, in some forms the dropletcontaining the initiator sequence will sequentially increase in volumewith the addition of each reagent droplet throughout the synthesisprocess.

b. Incubation Conditions

The methods employ different conditions to achieve synthesis ofbiopolymers. In preferred forms, the sequence of splitting, moving andcombining fluid droplets is interspersed with incubation periods tosynthesize a biopolymer through cycles of steps (1)-(6), above. Theincubation conditions can include changes to one or more parameters.Therefore, in some forms, incubation periods include changing ormanipulating one or more physical or chemical parameters, such astemperature, ionic concentration, pH, pressure, charge, exposure tolight, etc. In a preferred form, incubation conditions are used tocontrol the attachment of a component building block to an initiator,for example, to enhance or optimize, or reduce or prevent theattachment.

In some forms, the methods include specifying optimal conditions forattachment of each component building block. Therefore, parameters ofthe droplet can be varied, including volume, concentration etc., andexternal parameters, including incubation time, temperature, etc. can bevaried to control, optimize or minimize one or more aspects of theassembly process.

Exemplary incubation conditions include the conditions that produce themost effective results, as determined by the goal of the step of movingdroplet, combining two or more droplets, or splitting a droplet. In anexemplary form, the goal of combining an attachment reagent with aninitiator or a biopolymer and a component building block is optimized byenhancing the attachment of a single component building block to theinitiator or biopolymer. Therefore, optimal conditions include thosewhich most effectively achieve the attachment. Exemplary steps that canbe optimized include optimal conditions for catalysis of attachment(“attachment conditions”), optimal conditions for stopping or blocking areaction (“stop conditions”, and “blocking conditions”), and optimalconditions for rinsing, dissolving or washing reagents (“washconditions”). Typically, parameters that can be varied for each set ofconditions include (i) incubation volume, (ii) incubation time, and(iii) other conditions, such as those external from or independent ofthe droplet. Each of these parameters can be optimized by one skilled inthe art.

i. Incubation Volume

The methods include mixing of droplets of different sizes, or the samesize. Therefore, the methods can vary the amount and concentration ofthe reagents after combination of two of more droplets (i.e., the“final” concentration).

In an exemplary form, a volume of a buffer, or attachment reagent issplit from the corresponding reservoir and moved to combine with adroplet containing an initiator sequence, or a biopolymer, or a beadwith the initiator sequence, or biopolymer bound thereto, in an amountsufficient to produce a desired concentration in the resulting droplet.For example, a droplet can be increased in size until a desiredconcentration of reagent(s) is reached. In some forms, a dropletincluding an active agent is combined with a droplet containing noactive agent, such as a buffer or water droplet, to dissolve the activeagent and/or reduce the concentration to a desired value. This dropletis subsequently combined with a droplet containing an initiatorsequence, or a biopolymer. In this manner, the methods enable theuser-defined creation of droplets of specified volume having a specifiedconcentration of reagent(s), pH, ionic strength, etc. Therefore, in someforms, the methods include the step of creating droplet having a definedconcentration, pH, salt concentration, amount of active agent, etc.prior to combining with the droplet containing an initiator sequence, ora biopolymer. In this manner, specific concentrations of reagents can becombined with the addressed biopolymer throughout the assembly process,for example, to control the rate and extent of attachment of a givenbuilding block, or to block enzyme activity.

In an exemplary form, the concentration of a component building blockwithin a droplet is reduced such that only one or more such componentbuilding block are added to the initiator sequence, or terminal end ofthe biopolymer per cycle. Therefore, in some forms, the concentration ofthe component building block in the combined droplet determines thenumber of component building blocks that is added to the biopolymer percycle.

In another form, the concentration of salt or pH in the combined dropletis used to control enzyme activity. For example, the amount of salt andpH in a droplet can effect the rate and fidelity of an enzyme-catalyzedaddition reaction. Therefore, in some forms, droplets including acatalyst are combined with droplets including an amount of salt or asalt-free buffer sufficient to reduce or increase the salt concentrationin the combined droplet such that the activity of an enzyme catalyst isreduced, increased, prevented or initiated. For example, in some formsthe concentration of salt within the combined droplet is increased to anamount effective to initiate the activity of a catalyst. In other forms,the concentration of salt within the combined droplet is reduced to anamount effective to prevent the activity of a catalyst.

Typical incubation volumes are volumes between about 0.1 Picoliter (pl),and about 100 microliters (μl) (but can be larger), for example, betweenabout 1 pl and about 50 nanoliters (nl). In an exemplary form, eachdroplet size is between about 0.5 nl and 5 nl.

ii. Incubation Time

The methods include combining droplets to form a larger combined dropletat a given location for a specific period of time. After two or moredroplets are combined, they can be split, for example, to produce alarge droplet of solvent and a smaller volume that includes theimmobilized biopolymer, after a certain time period, for example toisolate the biopolymer form attachment reagents.

Therefore, in some forms the methods combine reagents for a specificperiod of time, for example, sufficient to achieve the goal of thecombining step. Exemplary incubation times include one or moremilliseconds (ms), one or more seconds, for example, 5 seconds, 10seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1hour, 90 minutes, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours or morethan 24 hours. In some forms, the incubation time is determinedaccording to the specific reactivity of the enzyme, reagent or catalystthat is required. For example, in some forms, the amount of time anattachment agent is incubated with an initiator or biopolymer and one ormore component building blocks is varied to limit the number ofcomponent building blocks that are attached. In an exemplary form, twoof more droplets of reagents are combined for a period of time between30 seconds and 5 minutes. An exemplary incubation time for catalysis ofattachment of a nucleotide component building block to a nucleic acid bythe TdT enzyme is 10 minutes at 37° C.

iii. Other Conditions

The methods include mixing of droplets under different conditions toachieve optimal incubation parameters. Therefore, the methods can varythe conditions under which the reagents are combined, for example, toprovide different amounts of heat, light, gas, electric charge, etc. Insome forms, incubation is enhanced by mixing the combined droplets, forexample by agitation of the support surface. An exemplary temperaturefor incubation of droplets for enzymic attachment is between 20° C. and40° C., for example 37° C. An exemplary temperature for reducing orpreventing the activity of a catalyst enzyme is a temperature greaterthan 40° C. for example, a temperature between 60° C. and 80° C. Thetemperature at a given location during the synthesis of a biopolymer canbe controlled, for example, by a Peltier temperature control system. Insome implementations the droplet is moved to a location on the grid thatcan be held at 37° C. from 1 second to 30 minutes, or for example 10minutes. In another implementation, a mobile heat block can be moved inthat sits at the base of the microfluidic channels that heat thechannels to 37° C., or the desired operating temperature. In anotherimplementation, the device is placed in a room that operates at 37° C.or the desired operating temperature.

c. Inhibition of Catalyst Activity

In some forms, the methods include the step of inhibiting the catalystactivity. Inhibiting the catalyst can include a process that reduces orprevents the addition of a component building block onto the biopolymer.Inhibiting the catalyst activity can be achieved by means includingactive inhibition of the catalyst enzyme; removal, or reduction in theamount of, one or more essential enzyme co-factors; removal, orreduction in the amount of, one or more component building blocks;disruption or degradation of the catalyst enzyme; physical separation ofthe biopolymer from the catalyst enzyme; and combinations of these.Therefore, in some forms, the methods inhibit the activity of thecatalyst by combining the droplet including the biopolymer with one ormore droplets including a reagent or molecule that inhibits or reducesthe activity or presence of the enzyme.

In some forms the methods inhibit the activity of the catalyst bycombining one or more inhibitory molecules into the biopolymer. Theinhibitory molecules can reversibly block the incorporation ofsubsequent component building blocks onto the biopolymer. Therefore, insome embodiments, the methods coordinate the sequence-specific synthesisof biopolymers by employing a sequence of steps to (i) activate orcombine, (ii) inhibit or remove, and (iii) re-activate or recombine thecatalyst enzyme. In some forms, the step of activating the catalyst (forexample, in the presence of a first component building block) includesone or more processes such as combining droplets including enzymeco-factors, buffers, or other reagents necessary for catalyst function.In some forms the activation step incudes incubating the combineddroplet, for example, for a specified time, at a specified temperature,etc. In some forms the step of inhibiting the catalyst includes one ormore processes such as combining droplets with reagents that chelate,sequester or otherwise remove the enzyme co-factors, buffers, or otherreagents necessary for catalyst function. In some forms the inhibitionstep incudes incubating the combined droplet, for example, for aspecified time, at a specified temperature, etc. to ensure the activityof the catalyst is inhibited. In some forms the step of reactivating thecatalyst (for example, in the presence of a second component buildingblock) includes one or more processes such as combining droplets withreagents including enzyme co-factors, buffers, or other reagentsnecessary for catalyst function.

In some forms the inhibition step includes the addition of one or moreinhibitory component building blocks to the biopolymer, for example, aninhibitory nucleic acid that includes a charged moiety which stericallyhinders the activity of the catalyst enzyme. Therefore, in some forms,the step of reactivating the catalyst activity includes removal of thecharged moiety from the inhibitory nucleotide.

In some forms, the sequence of (i) activating or combining the catalyst,(ii) inhibiting or removing the catalyst, and (iii) reactivating orrecombining the catalyst include one or more wash steps. For example, insome forms, one or more wash steps are carried out between (i) and (ii),between (ii) and (iii), between (i) and (iii), or between each of (i),(ii) and (iii).

In an exemplary form, the component building blocks are all inhibitorynucleic acids. Therefore, in some forms, every step for the addition ofa component building block to the biopolymer includes (i) and (iii),above. For example, the step of reactivating the catalyst includesremoval of the inhibitory moiety from the previously added nucleic acid.

In an exemplary form, the method employ a stop reagent that is achelating agent that removes cations from the solution containing thecatalyst enzyme. Therefore, in some forms the methods combine the use oflimiting concentrations of catalysts and/or component building blockswith chelating agents to provide precise control over the number ofcomponent building blocks that is added to a biopolymer at each “cycle”,for example to incorporate one, two, three, or four component buildingblocks to the growing biopolymer. Therefore, in some forms, the methodsinclude stop reagents that provide precise control over the length andsequence of the biopolymers that are synthesized. Therefore, in someforms, the methods do not produce biopolymers having a range of sizesand sequences according to a binomial distribution.

3. Exemplary Methods

Exemplary methods for the microfluidic device-based synthesis ofuser-defined nucleic acids are provided. The exemplary methodssynthesize nucleic acids in a highly parallel manner using template freeenzymatic synthesis of DNA by using the addition of nucleotides,enzymes, washing solution, and blocking solutions through programmedmovement with droplet-based microfluidic device technology. Theexemplary methods define the sequences of movement and parametersrequired for template-free assembly of nucleic acids using TdT enzyme asan attachment agent. The exemplary methods employ grid-based EWOD as onemethod of droplet technology, but can be generalized to discretegrid-based movement of droplets by any applied potential, such asthrough circuits or through optics, or any continuous induced movementof droplets from such a system. Therefore, the exemplary methods can begeneralized for use with any system that employs droplets of 1 pL, up to1 μL to be split, merged, or mixed. The exemplary methods employ dNTPs(for example, ATP, UTP, GTP, and CTP) as component building blocks foruser-defined nucleic acid sequences. The methods can be used to attachany bases known to the art that are recognized and can be attached byTdT polymerase.

Exemplary methods include (a) EWOD-based Synthesis of Nucleic Acid onsolid support; (b) EWOD-based Synthesis of Nucleic Acid usingimmobilized TdT enzyme; (c) EWOD-based Synthesis of Nucleic Acid inSolution; and (d) encoding data within biopolymer sequences, areprovided below.

a. EWOD-Based Synthesis of Nucleic Acid on Solid Support

In an exemplary method, EWOD-based synthesis is employed fortemplate-free synthesis of a user-defined nucleic acid, using aninitiator sequence specific for the Terminal deoxynucleotidyltransferase (TdT) polymerase enzyme coupled to a magnetic bead.

When deoxyribonucleotides polymerize to form DNA, the phosphate groupfrom one nucleotide will bond to the 3′ carbon on another nucleotide,forming a phosphodiester bond via dehydration synthesis. New nucleotidesare always added to the 3′ carbon of the last nucleotide, so synthesisalways proceeds from 5′ to 3′. An initiator sequence for the TdT enzymeis attached to magnetically active beads or directly to a surface bybinding to the beads or surface modified with streptavidin. Theconcentration is generally between about 1 fmol and 100 picomole per 1pL droplet size, up to 5 nL droplet size, or larger, for example, up to1,000 nL.

The magnetic beads are held in place by the presence of a magnetexternal to the surface of the EWOD chip. The affixed DNA initiatorsequence is maintained in aqueous solution throughout the synthesis. Theaqueous solution can be any aqueous solution suitable for maintainingthe synthesized nucleic acid. Using programmed droplet movement offeredby EWOD, component building blocks are sequentially added to theimmobilized initiator sequence by movement of a droplet containing thedesired nucleotide. Exemplary dNTPs include canonical dATP, dTTP, dGTP,or dCTP, and non-canonical dNTPs. A droplet containing the selectedcomponent building block is split from the corresponding reservoir, andthen moved across the grid network to the location (address) of thefixed strand droplet. Upon contacting the droplet containing the fixedstrand, the combined droplets are mixed. In some forms, the incomingdroplet containing the dNTP component building block may also containbuffering and salt components for the reaction and additionally TdTenzyme. Alternatively, the TdT enzyme could be separately mixed with thestationary droplet before or after the addition of nucleotides.

After mixing of the nucleotides with the growing DNA polymer and withthe addition of buffer and enzyme, the addition of nucleotides to thegrowing affixed polymer to begin. The time for incorporation isgenerally from 1 second to 1 minute, and the number of additions ofnucleotides as a homopolymer to the affixed polymer is determined by (1)temperature, (2) time in total solution, (3) presence of blockingmoieties on the dNTP that was added, and (4) the amount of dNTP thatwere added to the total solution. In case 1, the temperature can bemodified from 4° C. to 98° C. which has an effect on enzymeincorporation rates. Current standard operating temperatures are 37° C.The time the affixed growing polymer is subjected to the dNTPs and/orenzyme is also a factor for number of incorporations. By incubating thepolymer with the enzyme and dATP (for example) for 1 minute at 37° C.,incorporation of 1 to 10 to 100 homopolymer A's would assemble to theaffixed polymer. The time that the affixed strand is subjected to thedNTPs can be controlled by removing and washing the fixed-positionpolymer away from the dNTPs. The presence of blocking nucleotides whichcan be modified at, for example their 2′ or 3′ position, canadditionally be used to limit the length of the growing polymer, whichcan be achieved by having these modified nucleotides in the dNTP mixitself, or have them in high concentration in an external droplet thatis moved into and mixed with the solution. Finally, the homopolymeraddition of dNTPs can be limited by the concentration of the dNTPs,wherein the droplet might contain 1 pmol of dATP (for example) added toa droplet containing 1 pmol of affixed polymer. Thus, addition of thenucleotides will diminish to nothing as they are incorporated, and thenumber of additions per homopolymer will have a Poisson distributionaround a single nucleotide incorporation.

Sequences of chosen length will finally be released by low-salt,heating, or cleaving with a nuclease-specific cut site incorporated 5′of the component initiation sequence (e.g., PstI), or will be amplifiedusing polymerase chain reaction (PCR) off the chip. Alternatively theDNA polymer will not be released from the bead or surface, but willremain bound for further processing.

Ease of subsequent sequencing of ssDNA can be achieved by prepending orappending the SMRTbell (PacBio) polymerase sequence to the 5′ or 3′ ofthe growing DNA strand, or the component initiation sequence fornanopore sequencing (Oxford Nanopore). This allows for direct sequencingthrough adaptation to already discovered methods of sequencing.

b. Exemplary Method for EWOD-based Synthesis of Nucleic Acid usingimmobilized TdT enzyme

In some forms, the template-free polymerase (e.g. TdT) is affixed to asolid support by biotin moieties or by cloning with streptavidin, orother methods of fixation known to the art. Furthermore, the enzyme(e.g. TdT) is additionally modified to enhance binding to the growingsingle-strand polymer, such as by cloning at the N-terminus orC-terminus a single-stranded DNA binding protein such as SSB, orzinc-finger domains. Thus the polymerase is affixed to a solid support(bead, surface) and the template is attached non-covalently to thetemplate-free polymerase by interaction with a second domain. Theaddition of the nucleotides will then catalyze the addition and allmethods applied in example 1 could be applied here for sequence controlof the growing polymer.

c. Exemplary Method for EWOD-based Synthesis of Nucleic Acid in Solution

Starting with a low-volume, high concentration of Enzyme and initiatorsequence, the addition of the dNTPs will be sequence-specified and in aconcentration such that depletion will be limiting with each addition.Therefore if dATP (for example) was added to the enzyme and polymer mixat a 1:1 concentration, the dATP would be depleted over additions with aPoisson distribution of 1 A added per polymer. After reaction depletion,for example in 1 min at 37 C, the next nucleotide would be added andmixed to the solution, also in 1:1 amounts. Thus a growing,sequence-controlled DNA polymer could be made without affixing to asolid support and without requiring washing or removal of dNTPs.

An example of the steps necessary, for the sequence of EWOD-basedloading, moving and incubating of fluid droplets to synthesize thenucleic acid sequence “A-T-C-G” on a solid support (e.g., magnetic bead)using EWOD technology on a device represented in FIG. 2 is set forth inTable 1, below.

For the sequence of movement described in Table 1, the chip representedin FIG. 2 is configured as follows: “A” contains buffer, salt (e.g.,NaCl), dATP, and TdT; “T” contains buffer, salt (e.g., NaCl), dTTP, andTdT; “C” contains buffer, salt (e.g., NaCl), dCTP, and TdT; “G” containsbuffer, salt (e.g., NaCl), dGTP, and TdT; “Buffer 1” contains a washbuffer; “Buffer 2” contains a second wash buffer; “Release” contains abuffer and/or components to release the polymer from the support. Thereis also a collection port to retrieve the polymers, and a waste port.Typically, the system is at 37° C. Many of the steps can be parallelizedfor efficiency, as allowed by EWOD technology.

TABLE 1 Exemplary system and sequence for movement of droplets on amicrofluidic platform rendered as a grid according to FIG. 2. SequenceFROM TO 1 Load “A” to A1 2 A1 A3 3 Load “T” to E1 4 A3 B3 5 B3 C3 6 C3B3 (mixing) 7 Incubate 1 minute (Add A) 8 Buffer load to A1 9 Bufferload to A3 10 E1 C1 11 B3 B7 12 B7 Waste 13 A3 B3 14 A1 A3 15 C1 A1 16B3 B7 17 B7 Waste 18 A3 B3 19 Load “C” to C1 20 A1 A3 21 B3 B7 22 B7Waste 23 A3 B3 24 Incubate 1 minute (Add T) 25 Buffer load to A1 26Buffer load to A3 27 B3 B7 28 B7 Waste 29 A3 B3 30 A1 A3 31 C1 A1 32 B3B7 33 B7 Waste 34 A3 B3 35 A1 A3 36 B3 B7 37 B7 Waste 38 A3 B3 39Incubate 1 minute (Add C) 40 Load “G” to G1 41 Buffer load to A1 42Buffer load to A3 43 B3 B7 44 B7 Waste 45 A3 B3 46 A1 A3 47 G1 A1 48 B3B7 49 B7 Waste 50 A3 B3 51 B3 B7 52 A1 A3 53 B7 Waste 54 A3 B3 55Incubate 1 minute (Add G) 56 Release buffer load to A5 57 A5 A3 58 A3 B359 Incubate 1 minute 60 B3 B6 61 B6 Collect port

In some forms the sequence of 61 steps of loading and moving droplets inand out of a fixed, growing polymer set forth in Table 1 is input as acomputer-readable program.

d. Encoding of Digital Information

In an exemplary form, methods for microfluidic device-basedtemplate-free synthesis of DNA include encoding of digital informationas the switch between a base type to another base type. For example, aseries of 5 As (“AAAAA”), where 5 is representative of any number 1, 2,3, or more than 3 and A is representative of any base, would berepresentative of a 0, and a subsequent series of 6 Ts (“TTTTTT”), where6 is representative of any number 1, 2, 3, or more than 3 and T isrepresentative of any other base, would be representative of a 1. Thus,in this specific instance, “AAAAATTTTTT” would be representative of thedigital equivalent of “01”.

4. Manipulation of Biopolymers

In some forms, the methods add, remove, or modify a subset of componentbuilding blocks within an existing biopolymer. For example, in someforms, the methods attach additional component building blocks onto abiopolymer. In other forms, the methods remove one or more of thecomponents of the biopolymer, for example, by degrading one or morecomponent building blocks. In other forms, the methods modify anexisting sequence within a biopolymer, for example, by modification ofone or more chemical moieties of an existing residue, or by substitutionof one component building block for another. In some forms, a biopolymeris manipulated by a combination of the addition of one or morecomponents of a biopolymer and removal of one or more components of abiopolymer.

Manipulation of biopolymers is carried out according to the describedmethods for microfluidic-based movement of droplets including a dropletcontaining the biopolymer that is to be manipulated. In some forms thebiopolymer is immobilized on the microfluidic system. In other forms,the biopolymer is present in solution, for example, present in one ormore fluid reservoirs on a microfluidic device (e.g., an EWOD chip). Insome forms the biopolymer is manipulated by substitution, removal, oraddition of one or more sequences corresponding to a molecular orsequence barcode.

a. Molecular Barcoding

Molecular or sequence barcoding is a method of identifying moleculesfrom within a pool of other molecules. Barcoding is used, for example,for sequencing identification in next generation sequencing with complexpools of DNA strands. Barcoding can also be implemented for cell-basedidentification and RNA identification in solutions where parsing thesequences and samples are important for downstream separation of thesamples. The synthesis of the DNA for barcoding is typically achieved bypre-synthesis of the sequence using methods known in the art, and thenligated to the sample of interest by DNA ligase.

i. Adding Barcodes to Biopolymers

In some instances, the synthesized sequence-controlled polymer is abarcode for the recognition of the bead or the material within the bead.In some instances the barcode sequence is representative of informationthat is kept in silico for the access of the information. In someinstances the DNA sequence is algorithmically generated and not kept onan external computer. In an exemplary method, a set of pre-designedorthogonal barcodes are used as a basis set for point mutations thateither (i) maintain orthogonality similar to the original barcode set or(2) vary from one orthogonal barcode to another orthogonal barcode in asingle, double, or greater than double mutations. In the exemplarymethod, a neighborhood of 10 barcodes are generated surrounding theoriginal barcode. In each nearest neighbor of the barcode, a singlepoint mutation or many point mutations are introduced such that themelting temperature between the mutated barcode and the capture reversecomplement are varied by a pre-specified amount (e.g., 5 degrees). Thus,in each stepwise addition of more mutations, the temperature of capturelowers by, for example 5 degrees (or 1 degree or 20 degrees, or morethan 20 degrees). Thus, the sequence of the barcode is changed andcapture can be controlled by varying the sequence of the barcode or thecapture strand.

In some forms, the molecular barcode that is varied in a neighborhood ofsequences is representative of a description of underlying data, such asthe amount of red that exists in a picture that is encoded by the DNAsequences that are encapsulated. For example, in some experiments, apicture of a red Ferrari is converted to DNA sequences through methodsknown in the art. The DNA strands are then encapsulated in silica, andthe bead is barcoded to represent that the picture contains a red car.However, other images contain only partially red objects, such as apicture of a pink dress, that is only sometimes referred to as red, andthus would have a barcode of the red neighborhood, but would containseveral point mutations compared to true red. In other cases, thepicture may contain no red, such as a picture of a blue sky. In suchcases, the bead may not have a red barcode, or may have a barcode withenough mutations to render it “not red.” That picture may also thencontain a “100% Blue” barcode. Exemplary values that can be identifiedusing a corresponding nucleic acid barcode are presented in Table 2,below. Sequences in Table 2 represent twenty sequences that form a“neighborhood” of point mutations around the nucleic acid sequenceCGGCCCATCTGGTGTGATGCATTAC (SEQ ID NO: 1). In some forms, the sequencesof SEQ ID Nos. 2-21 in Table 2 represent an exemplary barcode “hash” forSEQ ID NO: 1.

TABLE 2 Exemplary Sequence Barcodes and  corresponding values MetadataBarcode associated Seq ID Number value Nucleotide Barcode No.  1100% Red CGGCCCATCTGGTGTGATGCATTAC Seq ID  No. 1  2  90% RedCGGCCCATCTGGTGTGATGCAGTAC Seq ID  No. 2  3  80% RedCGGCCCAACTGGTGTGATGCAGTAC Seq ID  No. 3  4  70% RedCGGCCCAACTGGTGTCATGCAGTAC Seq ID  No. 4  5  60% RedCGGTCCAACTGGTGTCATGCAGTAC Seq ID  No. 5  6  50% RedCGGTCCAACTGGTGTCAGGCAGTAC Seq ID  No. 6  7  40% RedCGGTCAAACTGGTGTCAGGCAGTAC Seq ID  No. 7  8  30% RedCAGTCAAACTGGTGTCAGGCAGTAC Seq ID  No. 8  9  20% RedCAGTCAAACTGGTCTCAGGCAGTAC Seq ID  No. 9 10  10% RedCAGTCAAACAGGTCTCAGGCAGTAC Seq ID  No. 10 11   0% RedCAGTCAAACAGTTCTCAGGCAGTAC Seq ID  No. 11 12 100% BlueGGCCAGATTATATGAGCGTCTCCTT Seq ID  No. 12 13  90% BlueGGCCAGATTATATGAACGTCTCCTT Seq ID  No. 13 14  80% BlueGGCCACATTATATGAACGTCTCCTT Seq ID  No. 14 15  70% BlueGGCCACATTAGATGAACGTCTCCTT Seq ID  No. 15 16  60% BlueGGCCACATTAGATGAACCTCTCCTT Seq ID  No. 16 17  50% BlueGGCAACATTAGATGAACCTCTCCTT Seq ID  No. 17 18  40% BlueGGCAACATTAGATGAACCTGTCCTT Seq ID  No. 18 19  30% BlueGGCAACATGAGATGAACCTGTCCTT Seq ID  No. 19 20  20% BlueGTCAACATGAGATGAACCTGTCCTT Seq ID  No. 20 21  10% BlueGTCAACATGAGATCAACCTGTCCTT Seq ID  No. 21

ii. Removing Barcodes of Biopolymers

In some forms, the barcodes are removed from a biopolymer. For example,if a biopolymer or bead includes a barcode, the sequence that includesone or more components of the barcode can be removed from the biopolymeror bead. In some forms the methods subsequently re-synthesize a newbarcode on the same biopolymer or bead. In some forms, the methodsinclude a sequence of steps for re-barcoding of a biopolymer or bead.Therefore, automated microfluidic-based methods for re-barcoding abiopolymer or bead are provided. In such cases, one or more barcodes areremoved from the biopolymer or bead.

Exemplary steps for removal of one or more component building blocksfrom a biopolymer include enzymatic cleavage or degradation, preferablyat one or more sequence-specific sites within the biopolymer. In anexemplary form, one or more nucleotides are removed from a biopolymer bythe activity of a nuclease enzyme, such as an exonuclease, orrestriction enzymes, or RNases that degrade the material of the barcode.In some forms, one or more amino acids are removed from a polypeptidesequence by a protease enzyme. In some forms, one or more componentbuilding blocks are removed from a biopolymer using chemistries thatdestabilize the molecule, such as a high pH (>10), for example, toremove RNA tags. In some forms the methods include one or more steps towash away the degrading or cleaving enzyme, or to remove thechemically-destructive factor from the biopolymer. In some forms themethods include one or more steps to synthesize a new barcode onto thebiopolymer.

In some forms, the methods further include removing or neutralizing theinhibitor in order to facilitate further nucleotide incorporation.Finally, nucleotides that are incorporated into a biopolymer can bedetectably labeled to monitor incorporation.

b. Encapsulation

In some forms, the methods encapsulate biopolymers. For example, in someforms, the methods include an additional step of encapsulating orotherwise covering a biopolymer in one or more outer layers. The outerlayers can be any material that is useful for the encapsulation of abiopolymer. Exemplary encapsulation materials include gels, silicates,lipids, proteins, oils, polymers and combinations of these. Reversibleencapsulation of nucleic acids in silica is describe in Paunescu, etal., Nature Protocols, volume 8, pages 2440-2448 (2013).

Synthesis of a biopolymer including the step encapsulation can enhancethe stability of the biopolymer. In an exemplary form, a biopolymer is anucleic acid sequence encoding one or more pieces of discrete data, forexample, bit-stream data. Encapsulation of data-sequences protects thedata-sequence from interrogation by other DNA sequences, in addition toadding thermal and chemical protection to the DNA.

In some forms, the encapsulated biopolymers are manipulated followingencapsulation. For example, in some forms the protected DNA are barcodedusing molecular recognition sequences such as biochemical tags andoptical signatures. These identifying barcodes can be used to segregatethe encapsulated data for retrieval and subsequent readout andconversion back to digital information.

Encapsulation or re-encapsulation of biopolymers can be carried outusing methods and materials known in the art. In some methods the wellor solution or synthetic cell-like compartment contains silica and allprecursors for optical barcoding with quantum dots, or calcium alginate,or polyacrylamide, or PEG or PEI, or other polymers typically used inthe formation of mineralized or hydrogel encapsulation. The catalyst forencapsulation will then be additionally added for the formation of nano-to micro-scale mineralized or hydrogel beads that encapsulate theinternal contents of the synthetic cell compartment or the well, or thedroplet in oil as implemented in the microfluidic device.

Typically, biopolymers having a sequence of any desired length arepackaged, encapsulated, enveloped, or encased in gel-based beads,protein viral packages, micelles, mineralized structures, siliconizedstructures, or polymer packaging, herein referred to as“sequence-controlled polymer objects”. In some forms, the synthesizedbiopolymers consist of a single, continuous polymer, contained within anencapsulation particle having nanometer dimensions. In some forms, thebiopolymers consist of many such polymers that are combined to becontained together within a single encapsulation particle. Thesediscrete biopolymer “packages” allow incorporation of one or morespecific molecular “tags” (such as barcodes) on the surface of thestructures. Some exemplary tags include nucleic acid sequence tags,protein tags, carbohydrate tags, and any affinity tags.

In some forms, the encapsulated particle will be barcoded or tagged by amolecular identifier such as an RNA, DNA, Locked nucleic acid, peptidenucleic acid, or peptide or protein or sugar or other recognitionpolymer that can be used to identify the particle by molecularinterrogation. In some instances, this identifier may be an antibody. Inother instances, this identifier may be a sequence specific polymer suchas a sequence of DNA. In some implementations this may be synthesizedusing the techniques described above by using a template free polymeraseand sequence-controlled additions for the active synthesis of thenucleic acid barcode. In some implementations, this may be synthesizedby addition of a pre-synthesized primer using a ligase, or a templatefree polymerase, or through chemical addition of the pre-synthesizedprimer to the particle through methods known in the art. In some casesthe barcode can be sequence-controlled but specifically generated formolecular recognition such as for a RNA aptamer or fluorescent RNAaptamer such as the Spinach aptamer, or by other RNA aptamers that canbe identified by interactions with other proteins or RNAs.

When an encapsulating agent is used to completely encase a one or morebiopolymers, the one or more biopolymer sequences can be present eitherwithin the particle core, or associated with one or more encapsulatinglayers surrounding the core, for example, embedded within anencapsulating material. Any indices/affinity/barcode tags are typicallyexposed and accessible at the surface of the particle. For example, insome forms, the indices/affinity tags are added in such a manner as tobe embedded within or otherwise attached to the external surface of theparticles.

In some forms, a molecular tag or barcode may need to be removed oraltered dynamically in an automated and pre-defined way, or in an activeway with feedback from a user or computer for dynamic memory allocationand re-allocation. In some implementations, the barcode can be digestedby a DNase, exonuclease, or restriction enzyme. In some instances wherethe barcode is RNA, RNase A or RNase T1, or other RNases can be used forbarcode removal, or can be removed by the presence of high pH. In someinstances, where the barcode is a peptide or protein or antibody orprotein tag such as a polyhistidine tag, the barcode can be removed bypeptidase or proteinase enzymes, or through pH. In anotherimplementation targeted photo/UV-degradation may be used. In each case,the encapsulated product may be optionally purified from the removalsolution and residual debris for later use.

Nanometer to micrometer-scale beads synthesized from polymers orcompounds such as, for example, silicon dioxide, can be synthesized byflow chemistry and microfluidics approaches. Silica precursors andoptical barcodes, such as dyes, quantum dots, lanthanides, and/or colorcenters are mixed with solvent and catalyst, and agitated until silicaparticles form. In another implementation, a reservoir containing silaneprecursors with dyes and/or quantum dots, lanthanide emitters, or colorcenters is mixed with DNA memory with other chemical precursors, such ascatalyst and solvent, through flow injection through a fluid junction ina flow chemistry set-up. The mixed precursors are passed through aheater to allow for silica formation.

In some forms, silica cores are synthesized with DNA memory and opticalbarcodes by mixing the silica precursors, optical barcodes, and DNAmemory with surfactant to form water-in-oil droplets. Resulting dropletsare incubated at 65° C. until silica forms. Precise size control ofparticles can be achieved by controlling the size of the water-in-oilemulsion.

In other forms, silica precursors, DNA memory, and optical barcodes aremixed using an automated liquid-handling device wherein specific volumesare dispensed into specific wells in 96-, 384-, 1536-well plates. Afterthe precursors are added into the well-plates, the well-plates are mixedwith agitation to produce silica particles.

In another form, silica precursors, DNA memory, and optical barcodes aremixed using droplets on a microfluidic device, for example, usingEWOD-actuated movement of droplets.

In some forms, sequence-controlled polymers synthesized either using theapproach defined here, or using another approach are grouped together onthe EWOD or other microfluidics device. In some forms,sequence-controlled polymers are grouped together by mixing synthesizedor added strands, or are kept separate. In a typical workflow, thestrands that are mixed are associated either for their sequences or forthe purpose of encoding similar data or part of the same bitstreamsequence.

In some instances, the mixed strands will be encapsulated. Encapsulationof biopolymers for use in nucleic acid memory systems is described inInternational publication No. WO 2017/189914. In some forms, silicananoparticles can be pre-manufactured, or manufactured on themicrofluidics device. Biopolymers, such as DNA, can be added into thesilica by ion-pairing of the phosphate backbone with theammonium-functionalized surface of silica particles. Therefore, in someforms, the methods include the step of encapsulating biopolymers withinsilica. In some forms, the methods produce ammonium functionalizedparticles by preparing a silica core containing one or more agents, suchas dyes, quantum dots, lanthanide emitters, or color centers, atspecific concentrations for optical barcoding. In some forms, theoptically-barcoded silica core is functionalized, for example, byaddition of 3-(trimethoxysilyl)propyl-trimethylammonium chloride. Themethods adsorb biopolymers into the silica core by combining thebiopolymer with the silica core. The methods optionally add a furtherlayer of silica (e.g., a silica “shell” is added), for encapsulationusing tetraethoxysilane.

Silica cores can be prepared in large-scale through flow chemistry andmicrofluidics approaches. Therefore, in some forms, a reservoircontaining silane precursors with dyes and/or quantum dots, lanthanideemitters, or color centers is mixed with biopolymers (e.g.,bitstream-encoded nucleic acids), and with other chemical precursors,such as catalyst and solvent, through flow injection through a fluidjunction in a continuous-flow microfluidic system.

In some forms, fluid including combined precursors is passed through aheater to allow for silica formation. The methods purify silica cores,which are then and passed through another tube for DNA barcoding of thesilica.

In some forms, silica cores are synthesized with biopolymers (e.g.,bitstream-encoded DNA) and optical barcodes, for example, by combiningthe silica precursors, optical barcodes, and DNA memory with surfactantto form water-in-oil droplets. The methods the step of incubating theresulting droplets at a suitable temperature (e.g., 65° C.), forsufficient time to allow the silica to form.

In some forms, silica precursors, DNA memory, and optical barcodes aremixed using droplets on an electrowetting device.

In some instances, the solid support is on a bead that is itselfcomposed all or in part of sequence controlled polymers such as DNA. Inone such example, the solid support is a bead that contains DNAsequences that are either generated by the system in previous runs, orexternally generated using methods known in the art. In some cases, theaddition of nucleotides to the solid support bead is using all of themethods described here. In other cases, the bead is a solid support andthe additional nucleotides are added by incubation with ligases, orother template-free polymerases or chemically synthesized using standardand known chemistries to generate the nucleic acid or other sequence inplace.

DNA barcodes are attached to the surface through covalent approaches,for example (but not limited to) amide bond linkage usingN-hydroxysuccinimidyl esters, Michael addition through by sulfur groups,azide-alkyne cycloaddition, strain-release cycloaddition, or othercovalent attachment chemistries that are known in the art. In oneexample, silica containing DNA memory is coated with amine functionalgroups using 3-aminopropytriethoxysilane, 3-aminopropyltrimethoxysilane,or other chemical derivatives that introduce amine functional groupsthat are known in the art. Treatment with glutaric anhydride, succinicanhydride, or other ring anhydrides known in the art to theamino-functionalized silica introduces carboxylic acid functional groupAmino-modified DNA is then attached using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide,1-hydroxybenzotriazole (HOBt),hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-7-aza-benzotriazole (HOAt), ethyl2-cyano-2-(hydroxyimino)acetate, 4-N,N-dimethylamino pyridine (DMAP), orother activating reagents that are known in the art. In another example,bifunctional crosslinker succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is added to theamino-functionalized silica to introduce a maleimide functional group.DNA barcodes are then introduced via Michael addition using sulfhydrylgroups on DNA. In another example, amino-functionalized silica istreated with 1-akyne NHS ester or dibenzocyclooctyne (DBC) NHS ester tointroduce alkynyl groups on the surface of the silica. Azide-containingDNA is attached using Cu-catalyzed cycloaddition or strain-releasecycloaddition. Any “click”-type functional groups known in the art canbe used to attach DNA barcodes on silica.

In some forms, the encapsulated product can be barcoded again with thesame or different barcode sequence. This addition of a new barcode issynthesized by methods listed above. This new synthesis allows forrebarcoding the system, or a single object, or two objects, or more thantwo objects.

Each case of the barcoding, barcode removal, and re-barcoding, can beaccomplished on a microfluidic device where the solution is movingacross the bead or encapsulated product, or surface to allow for washingor monomeric additions to the product.

In some implementations the barcodes used as identifiers to theparticles are orthogonal to other particles containing the same ordifferent sets of sequences. In some cases, the barcodes are designed tohave minimal cross-talk between them and other barcodes and otherbarcode complementary sequences.

In some instances, the barcodes are error prone and may vary by 1, 2, 3,4, or more than 4 nucleotides from the user specified barcode. In someinstances, the barcodes may be specified to have 1, 2, 3, 4, or morethan 4 mutations from the initial barcode. In some implementations, thebarcodes are equated with meanings, such as representative of the colorred, or blue, or the year, or a geographic location. In some instances,the specified point mutations are representative of the measure ofbarcode representation, such as a measuring the representation of redfrom 1 to 10 as how exact the barcode sequence is to the originalsystem-orthogonal sequence. In some instances, the barcode representingthe color red and the barcode representing blue can be mutated by 1, 2,3, 4, or more than 4 point mutations to allow the red barcode to be moresimilar to the blue barcode. Thus the underlying polymer may bedescribed as a variation of the red to blue spectrum based on the amountof mutations from the pure red or pure blue associated barcodes.

In some implementations, the representative barcodes can bealgorithmically generated or can be associated by an external table ordatabase.

In some implementations, the representative barcodes can be extracted orpulled down based on the correctness compared to the original barcode.Thus a barcode sequence more similar to red would get pulled down with ared complementary sequence and a “blue-er” barcode could be pulled downwith a blue complementary sequence.

The algorithmic control of the orthogonality of the barcodes isgenerally applicable to barcoding any molecule used for sequencing,polymerase chain reaction, single-cell sequencing, or any applicationwhere fuzzy searches over molecular data are applicable.

In some forms, the complementary sequences to the barcodes are labeledwith a fluorescent moiety such as Cy5, Cy3, ROX, Atto, or otherfluorescent molecules on the 5′, 3′ or internally. In these cases, thecomplementary sequence to the barcode of interest will interact byWatson-Crick base pairing. Using methods described above by the EWODdevice, or by other microfluidics devices and channels, the pool ofbarcoded particles can be washed and the particles can be sorted byFACS, or microscope imaging, or other imaging platforms that wouldsubsequently allow for sorting. In one form, the fluorescent read from acamera could be used to track a certain tagged particle, that could thenbe segregated from the population by an optically controlled EWODdevice, or by separation by FACS based sorting of the particles. In allcases, barcodes may be dynamically altered on-the-fly to relabel oralter barcodes based on external requirements, using the precedingstrategies.

D. Purification of Biopolymers

The methods include purification of the assembled biopolymers.

Purification separates assembled biopolymers/encapsulated biopolymersfrom the substrates and buffers required during the assembly process.Typically, purification is carried out according to the physicalcharacteristics of biopolymers. For example, the use of filters and/orchromatographic processes (FPLC, etc.) is carried out according to thesize and structural properties of the biopolymers.

1. Isolating Biopolymers from the Microfluidic Device

In some forms, biopolymers are purified from the synthesis device usingaffinity chromatography, or by filtration, such as by centrifugalfiltration, or gravity filtration. In some forms, filtration is carriedout using an Amicon Ultra-0.5 mL centrifugal filter (MWCO 100 kDa).

In some forms, isolating and/or purifying biopolymers includesseparation of the newly-synthesized biopolymer from a solid supportmatrix. When a solid support matrix is employed to anchor or otherwisecontrol the initiator sequence throughout synthesis, the biopolymer iscleaved or otherwise separated from the solid support followingcompletion of synthesis. Removing the biopolymer from a solid supportcan be carried out according to methods generally known in the art. Forexample, in some forms, the biopolymer is designed to include one ormore cleavage enzyme recognition sequences for cleavage of thebiopolymer following synthesis. Biopolymers can be removed from a solidsupport during or after synthesis, or after purification, or after oneor more steps for post-purification modification of the biopolymer.

When an enzyme is used to cleave a biopolymer from a solid supportmatrix, the biopolymer can be designed to include a specific cleavageenzyme recognition sequence at or near the desired cut-site. In anexemplary form, the cleavage recognition sequence is within or near tothe initiator sequence. For example, in some forms the biopolymer is anucleic acid, and the cleavage enzyme is an enzyme that specificallycuts nucleic acid upon recognition of a nucleic acid sequence. Exemplaryenzymes for use in the methods include restriction endonuclease (RE)enzymes, such as blunt cutting RE and overhang-producing RE.

Following purification, biopolymers can be placed into an appropriatebuffer for storage, and/or subsequent structural analysis andvalidation. Storage can be carried out at room temperature (i.e., 25°C.), 4° C., or below 4° C., for example, at −20° C. Suitable storagebuffers include PBS, TAE-Mg²⁺ or DMEM.

2. Validation of Synthesized Biopolymers

In some forms, the methods include steps for the validation of thesynthesized biopolymers.

a. Sequence Determination

Methods for validating biopolymers include sequencing of biopolymers.Sequencing can be carried out before, or following one or morepurification steps. Compositions and methods for sequencing ofbiopolymers are known in the art. In some forms, biopolymers areengineered either during or after synthesis to include one or morereagents or functional molecules to facilitate sequencing. For example,blunt ends produced by blunt-cutting RE are compatible with universalsequence adapters. In some forms, sequencing adapters for use in thedescribed methods are universal adapters that bind to DNA fragmentsproduced by any blunt-cutting restriction endonuclease enzyme. Universaladapters are compatible with the blunt ended DNA fragments created byall blunt-cutting RE enzymes. In some forms, the adapters are compatiblewith any double stranded DNA fragment having a single base overhang. Forexample, universal adapters can have a single-base overhang that iscomplementary to a single base overhang that is common to a pool ofdouble stranded DNA fragments. In some forms, the universal adapters arecompatible with all DNA fragments having a single adenine.

Preferred universal sequencing adapters are “Y-shaped” adapters(Y-adaptors). Y adapters allow different sequences to be annealed to the5′ and 3′ ends of each nucleic acid in a library (Shin, et al., NatureNeuroscience 17, 1463-1475 (2014)).

In some forms, the sequencing adapters are ILLUMINA® Y-adaptors, pairedwith the dA tailing step, prevent concatamer formation, increase thesequenceable fraction of the library, and allows for paired-endsequencing. Use of ILLUMINA® Y-adaptors also enables incorporation ofdual-indexed barcodes during library amplification, which facilitateslarge-scale, inexpensive multiplexing. In some forms, the adaptersenable selective PCR enrichment of adapter-ligated DNA fragments.Preferably, sequence adapters can bind to a flow cell. Therefore, thesequence adapters enable the associated DNA fragments to be manipulatedthrough multiple applications for next generation sequencing.

In some forms, the methods include the step of nucleic acid sequencedetermination. The biopolymers can be sequenced according to sequencingmethods known in the art, for example, using techniques described inU.S. Patent Publication No. 2007/0117102, and U.S. Patent PublicationNo. 2003/013880. In general, methods for nucleic acid sequencedetermination include exposing the target nucleic acid to a primer thatis complementary to at least a portion of the target nucleic acid, underconditions suitable for hybridizing the primer to the target nucleicacid, forming a template/primer duplex.

b. Detection of Labels

In some forms, the methods include the step of detecting one or morelabels or detectable moieties incorporated into the biopolymer. Forexample, any suitable/appropriate detection method may be used toidentify an incorporated label (e.g., a labelled nucleotide analog),including radioactive detection, optical absorbance detection, e.g.,UV-visible absorbance detection, optical emission detection, e.g.,fluorescence or chemiluminescence. Single-molecule fluorescence can becarried out using a conventional microscope equipped with total internalreflection (TIR) objective. The detectable moiety can be detected on asubstrate by scanning all or portions of each substrate simultaneouslyor serially, depending on the scanning method used. For fluorescencelabeling, selected regions on a substrate may be serially scannedone-by-one or row-by-row using a fluorescence microscope apparatus (seeU.S. Pat. No. 5,445,934; and U.S. Pat. No. 5,091,652). Devices capableof sensing fluorescence from a single molecule include scanningtunneling microscope (STM) and the atomic force microscope (AFM).Hybridization patterns may also be scanned using a CCD camera (e.g.,Model TE/CCD512SF, Princeton Instruments, Trenton, N.J.) with suitableoptics (Ploem, CCD (Chase-Completed-Device) in Fluorescent andLuminescent Probes for Biological Activity Mason, T. G. Ed., AcademicPress, Landon, pp. 1-11 (1993), such as described in Yershov et al.,Proc. Natl. Aca. Sci. 93:4913 (1996), or may be imaged by TV monitoring.For radioactive signals, a phosphorimager device can be used (Johnstonet al., Electrophoresis, 13566, 1990; Drmanac et al., Electrophoresis,13:566, 1992; 1993). Other commercial suppliers of imaging instrumentsinclude General Scanning Inc., (Watertown, Mass. on the World Wide Webat genscan.com), Genix Technologies (Waterloo, Ontario, Canada; on theWorld Wide Web at confocal.com), and Applied Precision Inc. Suchdetection methods are particularly useful to achieve simultaneousscanning of multiple attached target nucleic acids.

III. Systems for Synthesis of Biopolymers

A. Computer Implemented Systems

The systems and methods provided herein are generally useful forpredicting the design parameters that produce a biopolymer having auser-defined sequence. In some forms, the parameters corresponding tothe desired form and the desired sequence are input using acomputer-based interface that allows for the sequence input process tobe carried out in a completely in-silico manner. For example, in certainforms, the methods are implemented in computer software, or as part of acomputer program that is accessed and operated using a host computer. Inother forms, the methods are implemented on a computer server accessibleover one or more computer networks.

FIG. 1 depicts the work flow of methods that can be implemented. In someforms, a user accesses a computer system that is in communication with aserver computer system via a network, i.e., the Internet or in somecases a private network or a local intranet. One or both of theconnections to the network may be wireless. In a preferred form theserver is in communication with a multitude of clients over the network,preferably a heterogeneous multitude of clients including personalcomputers and other computer servers as well as hand-held devices suchas smartphones or tablet computers. In some forms the server computer isin communication, i.e., is able to receive an input query from or directoutput results to, one or more laboratory automation systems, i.e., oneor more automated laboratory systems or automation robotics configuredto automate synthesis of biopolymers according to the described methods.

The computer server where the methods are implemented may in principlebe any computing system or architecture capable of performing thecomputations and storing the necessary data. The exact specifications ofsuch a system will change with the growth and pace of technology, so theexemplary computer systems and components should not be seen aslimiting. The systems will typically contain storage space, memory, oneor more processors, and one or more input/output devices. It is to beappreciated that the term “processor” as used herein is intended toinclude any processing device, such as, for example, one that includes aCPU (central processing unit). The term “memory” as used herein isintended to include memory associated with a processor or CPU, such as,for example, RAM, ROM, etc. In addition, the term “input/output devices”or “I/O devices” as used herein is intended to include, for example, oneor more input devices, e.g., keyboard, for making queries and/orinputting data to the processing unit, and/or one or more outputdevices, e.g., a display and/or printer, for presenting query resultsand/or other results associated with the processing unit. An I/O devicemight also be a connection to the network where queries are receivedfrom and results are directed to one or more client computers. It isalso to be understood that the term “processor” may refer to more thanone processing device. Other processing devices, either on a computercluster or in a multi-processor computer server, may share the elementsassociated with the processing device. Accordingly, software componentsincluding instructions or code for performing the methodologies of theinvention, as described herein, may be stored in one or more of theassociated memory or storage devices (e.g., ROM, fixed or removablememory) and, when ready to be utilized, loaded in part or in whole intomemory (e.g., into RAM) and executed by a CPU. The storage may befurther utilized for storing program codes, databases of genomicsequences, etc. The storage can be any suitable form of computer storageincluding traditional hard-disk drives, solid-state drives, or ultrafastdisk arrays. In some forms the storage includes network-attached storagethat may be operatively connected to multiple similar computer serversthat comprise a computing cluster.

1. Preparation of Libraries of Addressed Biopolymers

In some forms, biopolymer libraries are designed by automated methods.Automated design programs for generating uniquely addressed biopolymersallow for a diverse set of sequences to be made, towards the synthesisof a library of biopolymer for diverse applications. In an exemplaryform, libraries of biopolymers with diverse sequences are useful forapplications in memory storage, or applications for the analysis of agenome. For example, in some forms, a library or libraries ofbiopolymers can be constructed with the same or different labels, suchas capture tags or target sequences complementary to one or more targetmolecules.

a. High-throughput Production of Biopolymers and Modifications

Systems for the automated synthesis of libraries of biopolymersincluding different modifications can be implemented using automatedmethods. Typically, computational systems are applied to automatesequence designs of a diverse set of uniquely addressed biopolymers,such as nucleic acids. Generally, the high-throughput library generationof user-defined biopolymers is achieved via multiple automated steps.Automated design programs for synthesizing from hundreds to thousands ofbiopolymer sequences, such as nucleic acid sequences, allows for adiverse set of molecules to be made, towards the synthesis of librariesof sequences for diverse applications.

In some forms, the sequences of biopolymers to be synthesized are inputas a batch or set of sequences, for example, from a library or database.In other forms, the sequences of biopolymers are generated prior to orat the point of being input, for example, by a computational algorithm.An exemplary computational approach generates a set of biopolymers withspecific sequences, sizes, structural or functional properties. Forexample, the number of biopolymer sequences generated in silico is about10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 10⁶, 10⁷,or more than 10⁷.

In preferred forms, high-throughput methods for generation of tens,hundreds or thousands of biopolymers employ automated liquid handlers.For example, high-throughput methods employ liquid dispensers forproviding reagents as reservoirs to a surface for automated dropletsplitting, movement and combining. The automation of the methods caninclude providing reagents as reservoirs to designated locations on asuitable microfluidic device surface, such as an EWOD chip. Generally,automation is preferred for synthesizing libraries of biopolymers. Usingstocks of component building blocks, in combination with EWOD-mediatedautomated droplet movement, high-throughput combinatorial libraries ofbiopolymers are readily generated. In some forms, the volumes andconcentrations of the reagent reservoirs are taken into considerationwhen deciding on the plate format.

In preferred forms, the automated methods simultaneously coordinatemovement of droplets to synthesize more than ten biopolymers at a giventime. The high-throughput methods allow fast generation of any number ofbiopolymers as desired for a library, for example, one thousand, twothousand, three thousand, four thousand, five thousand, six thousand,seven thousand, eight thousand, nine thousand, ten thousand, twentythousand, thirty thousand, forty thousand, fifty thousand, one hundredthousand, one million, and more than one million user-defined sequencecontrolled biopolymers. In some forms, combinatorial libraries ofbiopolymers include variations in, size, sequence, and optionallymodifications, allowing for one thousand, one million, or more than onemillion sequences in a library synthesized according to the automatedmethods.

In some forms, the methods employ custom-designed microfluidic deviceplatforms, such as a chip including a custom-designed number of channelsand wells. Techniques for the isolation, purification, or modificationof biopolymers that are describe for single structures are applicable tohigh-throughput systems, typically via filtration and buffer exchange.In further forms, techniques such as rapid-run gel based assays,quantitative PCR (qPCR) and sequencing are used for amplification,structural analysis, and validation.

In some forms all of the parameters for a synthesis process aredetermined from the input sequences(s), for example, by a computerprogram. The program will provide a grid network, and assign sequencesto corresponding addresses on the grid. For example, each uniquesequence is assigned to a unique address on the computer-generated gridfor fluid movement. In some forms, the program will also provide thesequences and other parameters for each initiator, correspondingcatalysts, wash and block buffers. The amount, concentration and addressof each reagent reservoir is determined, as well as the sequence ofmovement required to synthesize each biopolymer.

B. Graphical User Interface

In a preferred set of forms a computer server receives input submittedthrough a graphical user interface (GUI). The GUI may be presented on anattached monitor or display and may accept input through a touch screen,attached mouse or pointing device, or from an attached keyboard. In someforms the GUI will be communicated across a network using an acceptedstandard to be rendered on a monitor or display attached to a clientcomputer and capable of accepting input from one or more input devicesattached to the client computer. In other forms, a phone interface canidentify, read and or run entered sequences.

In the exemplary form, the GUI contains a target sequence selectionregion where the user selects the parameters to be input. In thisexemplary system a target sequence is indicated by clicking, touching,highlighting or selecting one of the sequence, or subsets of sequences,that are listed. In preferred forms, the target sequence is selectedfrom a user-selected library. In some forms, the target sequence isselected and then customized to include user-defined features.Customization may include using any computer programs capable of suchfunctions. Other parameters relating to the target sequence, such aslength, molecular weight, overall size, charge, structure, etc.

In some forms, the GUI enables entering or uploading one or moresequences, such as libraries of nucleic acid sequences. For example, theGUI typically includes a text box for the user to input one or moresequences. The GUI may additionally or alternatively contain aninterface for uploading a text file containing one or more querysequences.

In forms that include both options, the GUI may also contain radiobuttons that allow the user to select if the target sequence will beentered in a text box or uploaded from a text file. The GUI may includea button for choosing the file, may allow a user to drag and drop theintended file, or other ways of having the file uploaded. Any of theparameters can be entered by hand to further customize

The GUI also typically includes an interface for the user to initiatethe methods based on the sequence(s) requested or other parameters. Theexemplary GUI form includes a submit button or tab that when selectedinitiates a search according to the user entered or default criteria.The GUI can also include a reset button or tab when selected removesthat user input and/or restores the default settings.

The GUI will in some forms have an example button that, when selected bythe user, populates all of the input fields with default values. Theoption selected by the example values may in some forms coincide with anexample described in detail in a tutorial, manual, or help section. TheGUI will in some forms contain all or only some of the elementsdescribed above. The GUI may contain any graphical user input element orcombination thereof including one or more menu bars, text boxes,buttons, hyperlinks, drop-down lists, list boxes, combo boxes, checkboxes, radio buttons, cycle buttons, data grids, or tabs.

In some forms, the described systems and methods for the automated,programmed enzymic synthesis of biopolymers using a microfluidic deviceare controlled through one or more systems, databases or other resourcesthat are implemented within Cloud computing. Cloud computing is aninformation technology paradigm that enables ubiquitous access to sharedpools of configurable system resources and higher-level services thatcan be rapidly provisioned with minimal management effort, for example,over the Internet. For example, in some forms, the sequence of one ormore biopolymers is selected from one or more databases accessed viacloud-based computing. In other forms, a general user interfaceinterfaces with one or more databases implemented through cloud-basedcomputing, for example, to design a synthesis or manipulation sequencefor a given biopolymer. For example, in some forms, data is input at acloud-based GUI specifying one or more biopolymer sequences, and theoutput includes one or more of a component initiation sequence, thelocations and amounts of each component building block, enzyme catalyst,buffers, stop or blocking reagents (each as uniquely addressed positionson a microfluidic device, such as an EWOD chip), and a sequence ofmovements and other intermediary steps (incubations, temperature, light,etc.) required for synthesis. The sequence of movements for droplets orfluid flow parameters can be output in any suitable format, for example,computer-readable code. Output can include some or all of theinformation required for synthesis or manipulation of one or severalbiopolymers. In some forms, the output provides sequences of movementfor simultaneous synthesis or manipulation of tens, hundreds, thousandsor tens of thousands of biopolymers on one or more microfluidic systems.Exemplary information that can be provided as databases (e.g.,cloud-based databases) include target biopolymer sequences, barcodesequences, component initiation sequences, and encoded bitstream data,for example, as implemented in nucleic-acid memory systems.

In some forms, cloud-based resources are accessed and implemented todirect manipulation of barcoded nucleic acids and/or memory objects.Therefore, in some forms, the methods employ cloud-based systems todesign, synthesize and alter barcodes for use in the preparation andaccess of nucleic acid memory storage systems. In some forms, themethods construct and/or degrade one or more sequence barcodes presenton a nucleic acid or memory object, according to one or more commandsentered via a graphical user interface. For example, computer-basedsystems can be used to provide the sequences of movements and otherparameters required to prepare databases of nucleic acid memory objects.Therefore, in some forms, systems and methods implement graphical userinterfaces to access and organize the databases. In some forms, the userinput requests access to one or more pieces of data stored within adatabase. The data request can be any format, for example, a request forone or more images, or one or more pieces of literature or data. Thesystems and methods can direct selection of one or more pieces of data,degradation of non-selected data, and/or reproduction of the selecteddata, according to the requirements of the user, for example, byproviding the sequence of movements and other parameters necessary toactuate a microfluidic device loaded with the corresponding library ofnucleic acid memory objects and other reagents.

IV. Compositions for Microfluidic Device-Mediated Biopolymer Synthesis

Biopolymers having a user-defined sequence, synthesized according to thedescribed methods are provided. Methods for template-free synthesis ofbiopolymers require reagents including initiator sequences, componentbuilding blocks, assembly catalysts, assembly buffers, wash buffers,stop-buffers and block buffers, as well as reagents for manipulation andpurification of the assembled biopolymer, including reagents forcleavage, sequencing and amplification of the biopolymer.

Compositions for synthesizing modified biopolymers are also described.The microfluidic device-based synthesis for assembling biopolymersaccording to the described methods can include one or more modifiedcomponent building blocks, such as non-naturally occurring derivativesand analogs. In some forms, the biopolymers are synthesized to includeone or more modified component building blocks. In other forms, thebiopolymers are modified by the addition of functional moieties on themicrofluidic device following synthesis. For example, in some forms,biopolymers are functionalized to include one or more molecules that arecapable of binding or otherwise interacting with one or more targetmolecules. Compositions for the microfluidic device-based synthesis,manipulation, and purification or amplification of biopolymers aredescribed in further detail below.

A. Microfluidic Devices for Biopolymer Synthesis

Microfluidic devices and systems for the distribution and movement ofsmall volumes required for synthesis are provided. Platforms foractuating splitting, movement, and combining of sub-microliter volumesof fluid as independent droplets can be employed for the describedmethods. Exemplary systems and devices include acoustic dropletdistribution such as the ECHO® 555 liquid handling device availablecommercially, volumetric displacement distribution such as the Mosquitopipette robot, or ink-jet type fluidic distributors. Additionally, thesynthesis may occur by flow across a chip, with microwells or syntheticcompartments used for synthesis.

In some forms, the microfluidic device uses acoustic droplet ejection(ADE) to actuate movement of fluids. In other forms, the microfluidicdevice uses electrowetting on dielectric (EWOD) to actuate fluidmovement. In further forms, the microfluidic device utilizesphoto-electrowetting to actuate movement. In some forms, themicrofluidics device utilizes a combination of different mechanisms forfluid handling/controlled fluid movement. Typically, the microfluidicdevice will be integrated with a computer to enable the automated,programmed control of the device. Systems and software forcomputer-mediated control of microfluidic devices are known in the art(see, for example, ECHO® Software Applications, commercially availablefrom Labcyte).

1. Electrowetting on Dielectric (EWOD) Devices

In some forms, growing biopolymer is immobilized at an addressedlocation on the EWOD chip. For example, in some forms, the componentinitiation sequence or the catalyst includes one or more sequencesdesigned to hybridize or otherwise bind to stationary-phase objects suchas magnetic beads, surfaces, agarose or other polymer beads. In otherinstances, the component initiation sequence or the catalyst includesone or more sites for conjugation to a molecule. For example, thecomponent initiation sequence or the catalyst can be conjugated to aprotein, or non-protein molecule, for example, to enableaffinity-binding of the initiation sequence or the catalyst, or of thesynthesized polymer. Electrowetting-on-dielectric (EWOD) actuationenables digital (or droplet) microfluidics where small packets ofliquids are manipulated on a two-dimensional surface. An exemplary EWODplatform is a chip, such as a microfluidic chip. EWOD chip liquiddroplet driving systems are described for use in methods for EWOD-basedsynthesis of biopolymers.

The EWOD chips actuate movement of fluid droplets, for example, byelectrifying one or more driving electrodes to direct movement of liquiddroplets to target positions. Therefore, the EWOD chip has thecapability of moving droplets from one addressed position to another bythe application of electric potential at a neighboring location.

In some forms, the electrowetting device employs channels and wells forthe controlled movement and combining of fluids from reservoirs alongthe channels in the chip.

In some forms, the electrowetting device is a chip using anall-electronic (i.e., no ancillary pumping) real-time feedback controlof on-chip droplet generation. Therefore, digital microfluidic systemsthat operate without carrier flows and preferably without anymicro-channels are described for use with the described methods.Typically, the movement of fluids is actuated by driving mechanismsacting on the droplets locally, i.e., on individual droplets. EWODdevices and methods of use thereof are known in the art, for example, asdescribed in WO 2006/005880, WO 2013/102011, WO 2016/111251, US2017/0326524 A1, U.S. Pat. No. 8,304,253 B2, U.S. Pat. No. 8,883,014 B2,U.S. Pat. No. 8,459,295 B2, U.S. Pat. No. 8,834,695 B2, U.S. Pat. No.9,266,076 B2, U.S. Pat. No. 9,169,573 B2, U.S. Pat. No. 9,539,573 B1,U.S. Pat. No. 9,005,544 B2, U.S. Pat. No. 9,808,800 B2, and in Gong, etal., Lab Chip.; 8(6): 898-906 (2008). EWOD devices for DNA manipulationincluding polymerase chain reaction, ligation, cloning, generation oflarger DNAs from smaller primers are described in Lin, et al., Journalof Adhesion Science and Technology, 26 (12-17): pp. 1789-1804; PMCID:PMC4770201 (2012); and Choi, et al., Annu. Rev. Anal. Chem. 5, pp.413-40 (2012)). Systems for electrowetting on dielectric microfluidicsusing chips for high-throughput EWOD applications are described in thereview article entitled Parallel processing of multifunctional,point-of-care bio-applications on electrowetting chips published by Fairin the annals of 14th International Conference on Miniaturized Systemsfor Chemistry and Life Sciences, pp. 2095-2097 (2010).

The systems and devices described by Fair utilize an electric fieldestablished in the dielectric layer to create an imbalance ofinterfacial tension if the electric field is applied to only one portionof the droplet, which forces the droplet to move. Droplets are usuallysandwiched between two parallel plates with a filler medium, such assilicone oil. Requirements for high throughput, point-of-caremicrofluidic chips that can process raw physiological samplesinclude: 1) low number of input/output (I/O) ports and on-chip reagentstorage; 2) flexible chip architecture for efficient use of fluidicprocessing elements; 3) programmable electronic control; 4) parallel ormultiplexed operation; 5) low cross-contamination to allow resourcesharing; and 6) scalability.

B. Addressed Biopolymers

Template-free synthesis of biopolymers according to the describedmethods can simultaneously produce from one up to several tens ofthousands of addressed biopolymers having user-defined sequences.Exemplary classes of biopolymers that can be synthesized using automatedmethods include nucleic acids (e.g., DNA, RNA) polypeptides (e.g.,proteins, peptidomimetics), oligosaccharides (e.g., carbohydrates),lipids, block co-polymers, and combinations of these (glycol-peptides,lipo-peptides, glycolipids, etc.).

The methods synthesize Biopolymers in the absence of a templatesequence. Rather, the desired sequence of the biopolymer is provided,for example, as computer-readable data, to coordinate the sequentialmovement of droplets to assemble the desired molecule. In some forms,the input sequence is user-defined. In other forms, the user can selectthe sequence and size of the biopolymer to be generated at random.

Input data for a polymer sequence is typically provided in a computerreadable format that is converted to from a non-computer readableformat. In some forms, input data is in the form of biopolymer sequencethat is converted (e.g., by computer software) to control movement ofdroplets for microfluidic device-based synthesis of an encodedbiopolymer sequence that is distinct to the input sequence. For example,in some forms, input data is in the form of a nucleic acid sequence thatincludes one or more sequences of genomic DNA or messenger RNA (mRNA),and the DNA or mRNA sequence is converted to control movement ofdroplets for microfluidic device-based synthesis of the polypeptidesequence corresponding to the translated genomic DNA or mRNA sequence.In other forms, input data is in the form of a polypeptide sequence thatis converted to control movement of droplets to actuate synthesis of thecorresponding nucleic acid coding sequence. In some forms, the input isin the form of bitstream data, which is converted to control movement ofdroplets to actuate synthesis of a corresponding biopolymer sequenceencoding the bitstream data.

Schemes, techniques, and systems for encoding data in the form of asequence, such as a biopolymer, are known in the art. The describedmethods can include the step of converting data into or encrypting datawithin the sequence of one or more biopolymers.

A non-limiting list of sequence-controlled biopolymers includesnaturally occurring nucleic acids, non-naturally occurring nucleicacids, naturally occurring amino acids, non-naturally occurring aminoacids, peptidomimetics, such as polypeptides formed from alpha peptides,beta peptides, delta peptides, gamma peptides and combinations,carbohydrates, block co-polymers, and combinations thereof.Sequence-defined unnatural polymers closely resemble biopolymers, suchas polymers incorporating non-canonical amino acids. e.g.,peptidomimetics, such as β-peptides (Gellman, S H. Acc. Chem. Res., 31,173-180 (1998)), peptide nucleic acids (PNA), peptoids orpoly-N-substituted glycines (Zuckermann, et al., J. Am. Chem. Soc., 114, 10646-10647(1992)), Oligocarbamates (Cho, C Y et al., Science, 261,1303-1305(1993), glycomacromolecules, Nylon-type polyamides, and vinylcopolymers.

In some forms, the methods employ microfluidic device-mediated movementof droplets for synthesis of uniquely addressed sequences of nucleicacids. In some forms, the methods employ microfluidic device-mediatedmovement of droplets for synthesis of uniquely addressed sequences ofpolypeptides. In some forms, the methods employ microfluidicdevice-mediated movement of droplets for synthesis of uniquely addressedsequences of carbohydrates. In other forms, the methods employmicrofluidic device-mediated movement of droplets for synthesis ofuniquely addressed biopolymers that contain two or more classes ofmolecules, such as glycopeptides, glycolipids, lipopeptides, etc., ormodified variants of nucleic acids, peptides or carbohydrates. Anexemplary modified peptide is a peptidomimetic, such as an α-peptidepeptidomimetic, a β-peptide peptidomimetic, a δ-peptide peptidomimetic,or a γ-peptide peptidomimetic, or combinations of these.

In some forms, the methods include providing a biopolymer sequence froma pool containing a multiplicity of similar or different sequences. Insome forms, the pool is a database of known sequences.

1. Nucleic Acid Biopolymers

In a preferred form, the methods employ microfluidic device-mediatedmovement of droplets for synthesis of uniquely addressed nucleic acids.One or more of the parameters of the nucleic acid, including nucleotidesequence, size, melting temperature, charge, conformation, etc. areuser-defined. Nucleic acids synthesized according to the describedmicrofluidic device-based methods can be from 2 nucleotides in length,up to 100,000 nucleotides in length. In preferred forms, synthesizednucleic acids have a sequence of greater than 100 nucleotides in length,up to 1,000, 2,000, 3,000, 4,000, 5,000, or 10,000 nucleotides inlength. In some forms, the microfluidic device-based methods synthesizeone or more nucleic acids of more than 10,000 nucleotides in length. Insome forms, the methods simultaneously synthesize multiple differentnucleic acids, for example, between 1 and 10,000 uniquely addressednucleic acids having the same or different sequences can be synthesizedat any given time. In some forms, the methods simultaneously synthesizemore than 10,000 uniquely addressed nucleic acids having the same ordifferent sequences, for example, up to 20,000, 30,000, 40,000, 50,000,60,000, 70,000, 80,000, 90,000, up to 100,000 nucleotides in length.

In certain forms information is contained within the nucleic acidsequence that is provided. Therefore, in some forms, discrete sets ofdata are rendered as sequences of nucleic acids, for example, in a poolor library of nucleic acids. In some forms, a pool of nucleic acidsequences ranging from about 100-1,000,000 bases in size is provided. Insome forms, the nucleic acid sequences within a pool of multiple nucleicacid sequences share one or more common sequences. When nucleic acidsthat are provided are selected from a pool of sequences, the selectionprocess can be carried out manually, for example, by selection based onuser-preference, or automatically.

In some forms, the input nucleic acid sequence is not the same sequenceas chromosomal DNA, or mRNA, or prokaryotic DNA. For example, in someforms, the sequence has less than 20% sequence identity to anaturally-occurring nucleic acid sequence, for example, less than 10%identity, or less than 5% identity, or less than 1% identity, up to0.001% identity. Therefore, in some forms, the nucleic acid sequenceprovided as input is not the nucleic acid sequence of an entire gene, ora complete mRNA. For example, in some forms the input sequence is notthe same sequence as the open-reading frame (ORF) of a gene. In someforms, the input sequence is not the same nucleic acid sequence as aplasmid, such as a cloning vector. Therefore, in some forms, the inputsequence does not include one or more sequence motifs associated withthe start of transcription of a gene, such as a promoter sequence, anoperator sequence, a response element, an activator, etc. In some forms,the input sequence is not a nucleic acid sequence of a viral genome,such as a single-stranded RNA or single-stranded DNA virus. In otherforms, the input sequence(s) are composed of the sequences of cDNAs,genes, protein sequences, protein coding open reading frames, orbiological sequences that together in a pool form a database ofbiological sequences.

2. Encapsulated Biopolymers

The described methods for microfluidic-based assembly of can encapsulatebiopolymers to produce discrete “objects” or “units” having a range ofdifferent structures. For example, in some forms, biopolymer objectsinclude a core particle, onto which one or more sequence-encodedbiopolymers is bound.

Binding of sequence encoded biopolymers to a particle core can beachieved using covalent or non-covalent linkages. In some forms, a coremolecule is coated or coupled to a molecule which is an intermediaryreceptor, for example, a binding site that is recognized by one or moreligands associated with the sequence encoded biopolymer.Sequence-encoded biopolymers can be coupled or hybridized to thereceptor-coated core molecule. In some forms, the polymer/coresubstructure is then coated with one or more encapsulating agents (i.e.,“molecular shelling”) to produce a coated biopolymer/core structure,which is then optionally coupled to one or more address labels. Bindingof address labels to a coated biopolymer/core particle can be achievedusing covalent or non-covalent linkages, or hybridization ofcomplementary nucleic acids. DNA barcodes linked to genetic featuresgreatly facilitate screening these features in pooled formats usingmicroarray hybridization, and new tools are needed to design large setsof barcodes to allow construction of large barcoded mammalian librariessuch as shRNA libraries. A framework for designing large sets oforthogonal barcode probes is described here. The utility of thisframework was demonstrated by designing 240,000 barcode probes andtesting their performance by hybridization. From the testhybridizations, new probe design rules were discovered thatsignificantly reduce cross-hybridization after their introduction intothe framework of the algorithm. These rules should improve theperformance of DNA microarray probe designs for many applications.

3. Barcodes and Labels

In some forms, biopolymers synthesized according to the methods caninclude one or more components that act as a barcode or label. Barcodesand/or labels can be used to identify, isolate, sort, organize, degrade,maintain, store, purify or otherwise characterize or manipulate thebiopolymer, or pool of biopolymers to which they are associated.Barcodes and labels can be selected from a wide variety of detectable,sortable or otherwise scorable molecules. Exemplary barcodes and labelsinclude sequence identifiers, such as nucleotide or amino acidsequences; capture tags; and dyes or other detectable molecules. In someforms, one biopolymer includes one or more barcode or label. Barcodes orlabels that can be used to capture the barcoded biopolymer for a pool ofsimilar biopolymers are provided. Barcodes or labels that can be used todetect, quantify or otherwise assay the presence or absence of thebiopolymer are provided. Barcodes or labels that enable the sorting ormanipulation of the associated biopolymers are also provided. In someforms, the barcodes permit sorting, selecting, ordering, degradation,synthesis and manipulation of the associate biopolymers usingmicrofluidic systems.

a. Sequence Identifiers In some forms, the biopolymers include sequenceidentifiers (i.e., indexing or “barcoding” regions). Sequenceidentifiers can identify a biopolymer upon further processing. Forexample, in the case of combining biopolymers, the different sequencescan be identified using different tags. Exemplary sequence identifiersinclude a nucleotide sequence of varying but defined length that isuniquely used for identification of one or more specific nucleic acids.

In certain forms, each biopolymer includes one or more unique sequencesof component building blocks which enables identification of eachbiopolymer. In some forms, the biopolymers include two or more sequenceidentifiers, for identification using a dual-index system.

The length of the sequence identifier can be adjusted according to theneeds of the user. For example, a length of 4 component building blocksis sufficient to produce up to 256 different sequences. Exemplarybarcode sequences are nucleic acid sequences of between 4 and 10nucleotides in length, inclusive. Preferably, the tag sequenceidentifiers differ by at least one nucleotide amongst all the differentsamples. An exemplary sequence identifier is 6 nucleotides in length.

An exemplary barcoded biopolymer is a nucleic acid encoding bitstreamdata including a nucleotide sequence that acts as a barcode to identifythe encoded data. A DNA barcode is a short DNA sequence that uniquelyidentifies a certain linked feature, such as nucleic acid sequenceencoding one or more genes, or pieces of metadata. Linking features toDNA barcodes of homogenous length and melting temperature (Tm) allowsexperiments to be performed on the features in a pooled format, withsubsequent deconvolution by PCR followed by microarray hybridization orhigh throughput sequencing. DNA barcode technology greatly improves thethroughput of genetic screens, making possible experiments that wouldotherwise be quite time-consuming or laborious. Numerous resources andsoftware tools are currently available for designing DNA microarraybarcodes/probes (see, for example, Nielsen et al. Nucleic Acids Res31:3491-3496 (2003); Rouillard, et al., Nucleic Acids Res 31:3057-3062(2003); Wang, et al., Bioinformatics 19:796-802 (2003); Hu, et al. BMCBioinformatics 8:350 (2007); and Markham et al., Methods Mol Biol453:3-31 (2008)).

DNA barcodes linked to genetic features greatly facilitate screeningthese features in pooled formats using microarray hybridization.Compositions of nucleic acid barcodes having distinct and detectableproperties are known in the art. Xu et al describe the generation andcharacterization of 240,000 barcode probes, and test their performanceby hybridization. Test hybridizations identified new probe design rulesthat significantly reduce cross-hybridization after their introductioninto the framework of the algorithm. These rules should improve theperformance of DNA microarray probe designs for many applications (Xu,et al., Proc Nall Acad Sci, 106 (7) 2289-2294 (2009)). Therefore, thedescribed methods for microfluidic-based synthesis of biopolymers canproduce barcoded nucleic acids including one or more barcodes that canbe used to select a distinct biopolymer, or pool of biopolymers, basedupon one or more of the sequence characteristics of the barcode.Exemplary characteristics that can be sued for the selection andisolation include thermal hybridization and melting temperature. Theapplication of melting temperature to select and isolate a pool ofbiopolymers based upon melting and hybridization characteristics isrepresented in the Examples.

In some forms, sequence identifiers (i.e., barcodes) are included withininitiator sequences. In other forms, the identifiers are attached to theinitiator or to the growing biopolymer during the synthesis. In anexemplary form, a sequence identifier is attached to an initiator, or toa growing biopolymer as a single, pre-assembled unit.

Molecular or sequence barcoding is a method of identifying moleculesfrom within a pool of other molecules. Barcoding is used for sequencingidentification in next generation sequencing with complex pools of DNAstrands. Barcoding can also be implemented for cell-based identificationand RNA identification in solutions where parsing the sequences andsamples are important for downstream separation of the samples. Thesynthesis of the DNA for barcoding is typically achieved bypre-synthesis of the sequence using methods known in the art, and thenligated to the sample of interest by DNA ligase.

Nanometer to micrometer-scale beads synthesized from polymers orcompounds such as, for example, silicon dioxide, can be synthesized byflow chemistry and microfluidics approaches. Silica precursors andoptical barcodes, such as dyes, quantum dots, lanthanides, and/or colorcenters are mixed with solvent and catalyst, and agitated until silicaparticles form. In another implementation, a reservoir containing silaneprecursors with dyes and/or quantum dots, lanthanide emitters, or colorcenters is mixed with DNA memory with other chemical precursors, such ascatalyst and solvent, through flow injection through a fluid junction ina flow chemistry set-up. The mixed precursors are passed through aheater to allow for silica formation.

In another implementation, silica cores are synthesized with DNA memoryand optical barcodes by mixing the silica precursors, optical barcodes,and DNA memory with surfactant to form water-in-oil droplets. Resultingdroplets are incubated at 65° C. until silica forms. Precise sizecontrol of particles can be achieved by controlling the size of thewater-in-oil emulsion.

In another implementation, silica precursors, DNA memory, and opticalbarcodes are mixed using an automated liquid-handling device whereinspecific volumes are dispensed into specific wells in 96-, 384-,1536-well plates. After the precursors are added into the well-plates,the well-plates are mixed with agitation to produce silica particles.

In another implementation, silica precursors, DNA memory, and opticalbarcodes are mixed using droplets on an electrowetting device. Forexample, nucleic acids can be modified to include proteins or RNAshaving a known function, such as antibodies or RNA aptamers having anaffinity to one or more target molecules. Therefore, the biopolymersdesigned and synthesized according to the described microfluidicdevice-based methods can be functionalized biopolymers.

Biopolymers synthesized according to the described microfluidicdevice-methods can include one or more functional molecules at one ormore locations on or within the polymer. In some forms, the functionalgroup is located at one or more termini. In other forms, the functionalmoiety is located within the biopolymer sequence at a distance fromeither terminus. In other forms, biopolymers include one or morefunctional moieties located within the sequence, and within one or bothtermini. When a biopolymer is modified to include two or more functionalmoieties, the functional moieties can be the same, or different.

Typically, biopolymers are modified by chemical or physical associationwith one or more functional molecules. Exemplary methods of conjugationinclude covalent or non-covalent linkages between the biopolymer and afunctional molecule. In some forms, conjugation with functionalmolecules is through click-chemistry. In some forms, conjugation withfunctional molecules is through hybridization with one or more nucleicacid sequences present on the biopolymer.

b. Capture Tags

In some forms, the sequence of a biopolymer includes a capture tag. Acapture tag is any compound that is used to separate compounds orcomplexes having the capture tag from those that do not. Preferably, acapture tag is a compound, such as a ligand or hapten, which binds to orinteracts with another compound, such as ligand-binding molecule or anantibody. It is also preferred that such interaction between the capturetag and the capturing component be a specific interaction, such asbetween a hapten and an antibody or a ligand and a ligand-bindingmolecule.

A preferred capture tag is biotin. In some forms, biopolymers includeone or more sequences of component building blocks that act as capturetags, or “Bait” sequences to specifically bind one or more targetedmolecules. For example, in some forms, overhang sequences includenucleotide “bait” sequences that are complementary to any targetnucleotide sequence, for example HIV-1 RNA viral genome.

Typically, targeting moieties exploit the surface-markers specific to agroup of cells to be targeted. Exemplary targeting elements includeproteins, peptides, nucleic acids, lipids, saccharides, orpolysaccharides that bind to one or more targets associated with cell,or extracellular matrix, or specific type of tumor or infected cell.Targeting molecules can be selected based on the desired physicalproperties, such as the appropriate affinity and specificity for thetarget. Exemplary targeting molecules having high specificity andaffinity include antibodies, or antigen-binding fragments thereof.Therefore, in some forms, biopolymers include one or more antibodies orantigen binding fragments specific to an epitope. The epitope can be alinear epitope. The epitope can be specific to one cell type or can beexpressed by multiple different cell types. In other forms, the antibodyor antigen binding fragment thereof can bind a conformational epitopethat includes a 3-D surface feature, shape, or tertiary structure at thesurface of a target cell.

Biopolymers and encapsulated biopolymer objects can include one or morefunctional sequences that can capture one or more functional moieties,including but not limited to single-guide- or crispr-RNAs (crRNA),anti-sense DNA, anti-sense RNA as well as DNA coding for proteins, mRNA,miRNA, piRNA and siRNA, DNA-interacting proteins such as CRISPR, TALeffector proteins, or zinc-finger proteins, lipids, and carbohydrates.In other forms, synthesized biopolymers are modified with naturally ornon-naturally occurring nucleotides having a known biological function.Exemplary functional groups include targeting elements, immunomodulatoryelements, chemical groups, biological macromolecules, and combinationsthereof.

In some forms, functionalized synthesized biopolymers include one ormore DNA sequences that are complementary to the loop region of an RNA,such as an mRNA. Synthesized nucleic acids functionalized with mRNAsencoding one or more proteins are described. In one exemplary case, asynthesized biopolymer can be functionalized with 1 or 2 or more nucleicacid sequences that are complementary to the loop region of an RNA, forexample an mRNA, for example an mRNA expressing a protein.

In some forms, biopolymers include one or more targeting elements, forexample, to enhance targeting of the synthesized biopolymers to one ormore cells, tissues or to mediate specific binding to a protein, lipid,polysaccharide, nucleic acid, etc. For example, for use as biosensors,additional nucleotide sequences are included in the synthesizedbiopolymers.

Exemplary targeting elements include proteins, peptides, nucleic acids,lipids, saccharides, or polysaccharides that bind to one or more targetsassociated with an organ, tissue, cell, or extracellular matrix, orspecific type of tumor or infected cell. The degree of specificity withwhich the synthesized biopolymers are targeted can be modulated throughthe selection of a targeting molecule with the appropriate affinity andspecificity. For example, antibodies, or antigen-binding fragmentsthereof are very specific.

Typically, the targeting moieties exploit the surface-markers specificto a biologically functional class of cells, such as antigen presentingcells. Dendritic cells express a number of cell surface receptors thatcan mediate endocytosis. In some forms, synthesized biopolymers includenucleotide sequences that are complementary to nucleotide sequences ofinterest, for example HIV-1 RNA viral genome.

Additional functional groups can be introduced to synthesizedbiopolymers for example by incorporating biotinylated nucleotides intothe synthesized biopolymers. Any streptavidin-coated targeting moleculesare therefore introduced via biotin-streptavidin interaction. In otherforms, non-naturally occurring nucleotides are included for desiredfunctional groups for further modification. Exemplary functional groupsinclude targeting elements, immunomodulatory elements, chemical groups,biological macromolecules, and combinations thereof.

Typically, the targeting moieties exploit the surface-markers specificto a group of cells to be targeted. Exemplary targeting elements includeproteins, peptides, nucleic acids, lipids, saccharides, orpolysaccharides that bind to one or more targets associated with cell,or extracellular matrix, or specific type of tumor or infected cell. Thedegree of specificity with which the synthesized biopolymers aretargeted can be modulated through the selection of a targeting moleculewith the appropriate affinity and specificity. For example, antibodies,or antigen-binding fragments thereof are very specific.

In some forms, biopolymers are modified to include one or moreantibodies. Antibodies that function by binding directly to one or moreepitopes, other ligands, or accessory molecules at the surface of cellscan be coupled directly or indirectly to the biopolymers. In some forms,the antibody or antigen binding fragment thereof has affinity for areceptor at the surface of a specific cell type, such as a receptorexpressed at the surface of macrophage cells, dendritic cells, orepithelial lining cells. In some forms the antibody binds one or moretarget receptors at the surface of a cell that enables, enhances orotherwise mediates cellular uptake of the antibody-bound biopolymers, orintracellular translocation of the antibody-bound biopolymer, or both.

Any specific antibody can be used to modify the nucleic acidbiopolymers. For example, antibodies can include an antigen binding sitethat binds to an epitope on the target cell. Binding of an antibody to a“target” cell can enhance or induce uptake of the associated nucleicacid biopolymers by the target cell protein via one or more distinctmechanisms.

In some forms, the antibody or antigen binding fragment bindsspecifically to an epitope. The epitope can be a linear epitope. Theepitope can be specific to one cell type or can be expressed by multipledifferent cell types. In other forms, the antibody or antigen bindingfragment thereof can bind a conformational epitope that includes a 3-Dsurface feature, shape, or tertiary structure at the surface of thetarget cell.

In some forms, the antibody or antigen binding fragment that bindsspecifically to an epitope on the target cell can only bind if theprotein epitope is not bound by a ligand or small molecule.

Various types of antibodies and antibody fragments can be used to modifynucleic acid biopolymers, including whole immunoglobulin of any class,fragments thereof, and synthetic proteins containing at least theantigen binding variable domain of an antibody. The antibody can be anIgG antibody, such as IgG1, IgG2, IgG3, or IgG4 subtypes. An antibodycan be in the form of an antigen binding fragment including a Fabfragment, F(ab′)2 fragment, a single chain variable region, and thelike. Antibodies can be polyclonal, or monoclonal (mAb). Monoclonalantibodies include “chimeric” antibodies in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey specifically bind the target antigen and/or exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; and Morrison, et al.,Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The antibodies canalso be modified by recombinant techniques, for example by deletions,additions or substitutions of amino acids, to increase efficacy of theantibody in mediating the desired function. Substitutions can beconservative substitutions. For example, at least one amino acid in theconstant region of the antibody can be replaced with a different residue(see, e.g., U.S. Pat. No. 5,624,821; U.S. Pat. No. 6,194,551; WO9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993)). In somecases changes are made to reduce undesired activities, e.g.,complement-dependent cytotoxicity. The antibody can be a bi-specificantibody having binding specificities for at least two differentantigenic epitopes. In one form, the epitopes are from the same antigen.In another form, the epitopes are from two different antigens.Bi-specific antibodies can include bi-specific antibody fragments (see,e.g., Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A., 90:6444-48(1993); Gruber, et al., J. Immunol., 152:5368 (1994)).

Antibodies that target the biopolymers to a specific epitope can begenerated by any techniques known in the art. Exemplary descriptions oftechniques for antibody generation and production include Delves,Antibody Production: Essential Techniques (Wiley, 1997); Shephard, etal., Monoclonal Antibodies (Oxford University Press, 2000); Goding,Monoclonal Antibodies: Principles And Practice (Academic Press, 1993);and Current Protocols In Immunology (John Wiley & Sons, most recentedition). Fragments of intact Ig molecules can be generated usingmethods well known in the art, including enzymatic digestion andrecombinant techniques.

c. Dyes or Other Detectable Labels

In some forms, biopolymers include one or more molecules that act as adetectable label or dye.

In some forms, the label is an optically-detectable moiety (e.g., afluorophore). Non-limiting examples of types of optically-detectablelabels include a fluorescent, chemiluminescence, or electrochemicallyluminescent label. Examples of fluorescent labels include, but are notlimited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid;acridine and derivatives thereof such as acridine, acridineisothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 15 1);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamine-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylaminolnaphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosanilin; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalocyanine; naphthalocyanine; any of the fluorescentlabels available from Atto-Tec, such as Atto 390, Atto 425, Atto 465,Atto 488, Atto 495, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590,Atto 594, Atto 610, Atto 611X, Atto 620, Atto 633, Atto 635, Atto 637,Atto 647, Atto 647N, Atto 655, Atto 680, Atto 700, Atto 725, Atto 740,etc.; any of the fluorescent labels available from Dyomics such asDY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, Dy-647, Dy-648,DY-649, Dy-650, Dy-651, DY-652, etc.; any of the fluorescent labelsavailable from Pierce such as DyLight 405, DyLight 488, DyLight 549,DyLight 633, DyLight 649, DyLight 680, DyLight 800, etc.; any of thefluorescent labels available from AnaSpec such as HiLyte Fluor™ 488dyes, HiLyte Fluor™ 555 dyes, HiLyte Fluor™ 647 dyes, HiLyte Fluor™ 680dyes, HiLyte Fluor™ 750 dyes, HiLytePlus™ 555 dyes, HiLytePlus™ 647dyes, HiLytePius™ 750 dyes, etc.; any of the fluorescent labelsavailable from Denovo Biolables such as Oyster 500, Oyster 550 P, Oyster550 D, Oyster 556, Oyster 645, Oyster 650 P, Oyster 650 D, Oyster 656,etc.; IRDye® 680, IRDye® 700, IRDye® 700DX, IRDye® 800, IRDye® 800 RS,IRDye® 800 CW, etc.; any of the fluorescent labels available from SETABiomedicals such as Seta K1-204, Seta K5-3212, Seta K8-1342, SetaK8-1352, Seta K8-1357, Seta K8-1407, Seta K8-1642, Seta K8-1644, SetaK8-1663, Seta K8-1664, Seta K8-1669, Seta K8-3002, Seta K4-1082, SetaK8-1669, Seta K7-545, Seta K7-547, Seta K7-549, Seta K8-1252, SetaK8-1261, Seta K8-1262, Seta K8-1320, Seta K8-1344, Seta K8-1367, SetaK8-1377, Seta K8-1382, Seta K8-1446, Seta K8-1667, Seta K8-1752, SetaK8-1762, Seta K8-1767, Seta K8-1777, Seta K8-1782, etc.

C. Substrates for Solid-Support Based Synthesis

Substrates for use as solid support matrices in methods for thetemplate-free synthesis of biopolymers are described. In some forms,capture tags incorporated into initiator sequences allow the initiatorsequence and growing biopolymer to be captured by, adhered to, orcoupled to a substrate. Such capture allows simplified washing andhandling of the biopolymers, and allows automation of all or part of themethod.

Capturing biopolymers on a substrate may be accomplished in severalways. In some forms, capture docks are adhered or coupled to thesubstrate. Capture docks are compounds or moieties that mediateadherence of a biopolymer by binding to, or interacting with, a capturetag on the fragment. Capture docks immobilized on a substrate allowcapture of the biopolymers on the substrate. Such capture provides aconvenient way of washing away reaction components that might interferewith subsequent steps.

Solid support substrates for use in the disclosed method can include anysolid material to which components of the assay can be adhered orcoupled. Examples of substrates include, but are not limited to,materials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, polysilicates,polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters,polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids.Substrates can have any useful form including thin films or membranes,beads, bottles, dishes, fibers, woven fibers, shaped polymers, particlesand microparticles. Some forms of substrates are plates and beads. Auseful form of beads is magnetic beads.

In some forms, the capture dock is an oligonucleotide. Methods forimmobilizing and coupling oligonucleotides to substrates are wellestablished. For example, suitable attachment methods are described byPease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method forimmobilization of 3′-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383(1995). A preferred method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic acids Res. 22:5456-5465(1994).

In some forms, the capture dock is the anti-hybrid antibody. Methods forimmobilizing antibodies to substrates are well established.Immobilization can be accomplished by attachment, for example, toaminated surfaces, carboxylated surfaces or hydroxylated surfaces usingstandard immobilization chemistries. Examples of attachment agents arecyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin,photocrosslinkable agents, epoxides and maleimides. A preferredattachment agent is glutaraldehyde. These and other attachment agents,as well as methods for their use in attachment, are described in Proteinimmobilization: fundamentals and applications, Richard F. Taylor, ed.(M. Dekker, New York, 1991), Johnstone and Thorpe, Immunochemistry InPractice (Blackwell Scientific Publications, Oxford, England, 1987)pages 209-216 and 241-242, and Immobilized Affinity Ligands, Craig T.Hermanson et al., eds. (Academic Press, New York, 1992). Antibodies canbe attached to a substrate by chemically cross-linking a free aminogroup on the antibody to reactive side groups present within thesubstrate. For example, antibodies may be chemically cross-linked to asubstrate that contains free amino or carboxyl groups usingglutaraldehyde or carbodiimides as cross-linker agents. In this method,aqueous solutions containing free antibodies are incubated with thesolid-state substrate in the presence of glutaraldehyde or carbodiimide.For crosslinking with glutaraldehyde the reactants can be incubated with2% glutaraldehyde by volume in a buffered solution such as 0.1 M sodiumcacodylate at pH 7.4. Other standard immobilization chemistries areknown by those of skill in the art.

D. Component Initiation Sequences

Methods for microfluidic device-based synthesis of biopolymers employinitiator sequences. An initiator sequence for use in the microfluidicdevice-based synthesis of biopolymers includes a recognition site for acatalyst. The initiator sequence will be selected according to class andcomposition of biopolymer that is to be synthesized.

In some forms, the initiator sequence is a component of the user-definedbiopolymer. In other forms, the initiator sequence is not a component ofthe user-defined polymer, but is removed following or during synthesis,for example, by exposure to one or more specific cutting enzymes.

In some forms, the component initiation sequence includes one or moresequences designed to hybridize or otherwise bind to solid support orstationary-phase objects such as magnetic beads, surfaces, agarose orother polymer beads. In other instances, the component initiationsequence includes one or more sites for conjugation to a molecule. Forexample, the component initiation sequence can be conjugated to aprotein, or non-protein molecule, for example, to enableaffinity-binding of the component initiation sequence, or of thesynthesized polymer.

In some instances, the initiator is biotinylated for capturing thebiopolymer on a streptavidin-coated bead. In some instances, theinitiator sequence is modified with chemical moieties. Non-limitingexamples include Click-chemistry groups (e.g., azide group, alkynegroup, DIBO/DBCO), amine groups, and Thiol groups. In some instancessome bases located inside a nucleic acid initiator sequence are modifiedusing base analogs (e.g., 2-Aminopurine, Locked nucleic acids, such asthose modified with an extra bridge connecting the 2′ oxygen and 4′carbon) to serve as linker to attach functional moieties (e.g., lipids,proteins). Alternatively DNA-binding proteins or guide RNAs can be usedto attach secondary molecules to the initiator sequence.

Exemplary component initiation sequences include nearly anysingle-strand DNA sequence longer than 2, 3, 4, or greater than 4nucleotides. In one example, the sequence GTCGTCGTCCCCTCAAACT (SEQ IDNO: 22) was used for initiation. In another example, the T7 promotersequence was used (TAATACGACTCACTATAG; SEQ ID NO: 23). In anotherpossibility, the sequence used for sequencing adapters could be used forinitiation such as, for example, the SmrtBell PacBio sequence(ATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT; SEQ ID NO: 24) or theinitiator sequence for Oxford Nanopore sequencing devices. In addition,other sequences may be used that include sites for nuclease andrestriction enzymes to function such as including a PstI cut site(CTGCAG) or EcoRI cut site (GAATTC).

1. Capture Tags

In some forms the initiator sequence includes one or more capture tags,for example, to couple the initiator/the growing biopolymer to a solidsupport matrix, or another molecule. Preferably, the capture tag is acompound, such as a ligand or hapten, which binds to or interacts withanother compound, such as ligand-binding molecule or an antibody. It isalso preferred that such interaction between the capture tag and thecapturing component be a specific interaction, such as between a haptenand an antibody or a ligand and a ligand-binding molecule.

A preferred capture tag is biotin. In an exemplary form, the initiatoris a biotinylated initiator. In a preferred form the biotinylatedinitiator is a biotinylated nucleic acid initiator.

In the disclosed method, capture tags incorporated into initiatorsequences allow the initiator to be captured by, adhered to, or coupledto a substrate, such as magnetic bead.

E. Component Building Blocks

Component building blocks that can be assembled into biopolymers aredescribed. The component building blocks can be any primary structuralunit that an initiator sequence for use in the microfluidic device-basedsynthesis of biopolymers includes a recognition site for a catalyst.

Exemplary recognition sequences include naturally-occurring nucleotides,amino acids, monosaccharides, lipids, as well as non-naturally occurringderivatives thereof.

1. Nucleotide Component Building Blocks

In some forms, the component building block is a deoxyribonucleotidemonomer (“nucleotide”). Nucleotide component building blocks can benaturally-occurring nucleotides, or non-naturally occurring derivatives.For example, when a nucleic acid sequence is synthesized, themicrofluidic device is loaded with one or more reservoirs including oneor more nucleic acids in a suitable buffer. Exemplary buffers includesterile filtered water and physiological saline.

Exemplary nucleotide component building blocks include, but are notlimited to the four standard nucleobases, adenine, guanine, cytosine,and thymine, as well as uracil, and modified variants thereof.

Reservoirs of nucleotide component building blocks can include a singlenucleotide species, or mixtures of two or more nucleotides. Whenreservoirs of nucleotides include mixtures, the relative amounts and/ormolar ratios of each nucleotide species can be varied according to thedesired compositions of the user-defined sequences to be synthesized. Insome forms, the reservoirs of nucleotides include oligomers of two ormore nucleic acids covalently linked by a phosphodiester bond.Incorporation of pre-determined oligomers of nucleotides as componentbuilding blocks can enhance the speed and efficacy of microfluidicdevice-based nucleic acid synthesis, reduce errors, include specificfunctionalized molecules, etc.

In some forms, the reservoir well contains one or more types ofnaturally occurring nucleotides, or one or more types of functionalizednucleotides, or mixtures, at a concentration at about 100 nM, 200 nM,300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 mM, or morethan 1 mM. For example, in certain forms, a droplet of 1 nL ofnucleotide component building blocks is split from a source wellcontaining nucleotide component building blocks at a concentration ofmore than 1 mM.

a. Modified Nucleotides

In some forms, the nucleotide component building blocks are “modified”nucleotides. Modified nucleotides include any non-naturally-occurringderivative of a naturally-occurring deoxyribonucleotide. When modifiednucleotides are to be incorporated into growing nucleic acidbiopolymers, the modified nucleotides can be present in a reservoir onthe microfluidic device (e.g., EWOD chip) as an independently addressedreservoir, or they can be mixed into a reservoir containing native(non-modified) nucleotides. For example, modified nucleotides can bemixed as a percentage or ratio of the total nucleotides within thereservoir. In some forms, the modified nucleotides represent 0.1% ormore than 0.1% of the total number of nucleotides in the reservoir, upto or approaching 100% of the total nucleotides in the reservoir,between 0.1% and 100% inclusive, such as 0.1%-0.5%, 1%-2%, 1%-5%,1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or more than 50% of thetotal, such as 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the total.

When modified nucleotides are used, they can be present in the same ordifferent regions of two or more simultaneously synthesized biopolymers.In some forms, synthesized biopolymers include the same or differentnumbers of modified nucleotides. In some forms, the modified nucleotidesare present at the equivalent position in every simultaneouslysynthesized biopolymer. Therefore, in some forms, a population ofsimultaneously synthesized nucleic acids include modified nucleotides atprecise locations and in specific numbers or proportions as determinedby the input sequence(s). In some forms, synthesized nucleic acidsinclude a defined number or percentage of modified nucleotides atspecified positions within the synthesized biopolymer. In some forms,synthesized nucleic acids produced according to the describedmicrofluidic device-based methods include more than a single type ofmodified nucleic acid.

Modified nucleic acid building blocks can be included to producestructural, and/or functional changes in a synthesized nucleic acidrelative to the equivalent non-modified form. In some forms, nucleicacid component building blocks are modified at the base moiety (e.g., atone or more atoms that typically are available to form a hydrogen bondwith a complementary nucleotide and/or at one or more atoms that are nottypically capable of forming a hydrogen bond with a complementarynucleotide), sugar moiety or phosphate backbone.

In some forms, nucleic acid component building block containamine-modified groups, such as aminoallyl-dUTP (aa-dUTP) andaminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment ofamine reactive moieties, such as N-hydroxy succinimide esters (NHS).

In other forms, nucleotide component building blocks include aphosphorothioate modified backbone to increase the stability of thesynthesized nucleic acid relative to non-modified nucleic acids, forexample, to protect against or reduce degradation by exonuclease.

Exemplary modified nucleotide component building blocks include, but arenot limited to, diaminopurine, S2T, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine, pyrazolo[3,4-d]pyrimidines, 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine.

In some forms, the nucleotide component building blocks are lockednucleic acids (LNA) or peptide nucleic acids (PNA).

i. Locked Nucleic Acids

In some forms, the component building blocks are locked nucleic acids(LNA). LNA is a family of conformationally locked nucleotide analogueswhich, amongst other benefits, imposes truly unprecedented affinity andvery high nuclease resistance to DNA and RNA oligonucleotides(Wahlestedt, et al., Proc. Natl Acad. Sci. USA, 975633-5638 (2000);Braasch, et al., Chem. Biol. 81-7 (2001); Kurreck, et al., Nucleic AcidsRes. 301911-1918 (2002)). In some forms, the nucleic acids are syntheticRNA-like high affinity nucleotide analogue, locked nucleic acids. Insome forms, the nucleotides are locked nucleic acids.

ii. Peptide Nucleic Acid (PNA)

In some forms, the component building blocks are peptide nucleic acid(PNA). PNA is a nucleic acid analog in which the sugar phosphatebackbone of natural nucleic acid has been replaced by a syntheticpeptide backbone usually formed from N-(2-amino-ethyl)-glycine units,resulting in an achiral and uncharged mimic (Nielsen P E et al., Science254, 1497-1500 (1991)). It is chemically stable and resistant tohydrolytic (enzymatic) cleavage. In some forms, the scaffolded DNAs arePNAs. In other forms, the nucleotide component building blocks are PNAs.In some forms PNAs, DNAs, RNAs, or LNAs are used for capture, orproteins or other small molecules of interest to target, or otherwiseinteract with complementary binding sites on structured RNAs, or DNAs.In other forms, a combination of PNAs, DNAs, RNAs and/or LNAs is used inthe microfluidic device-based synthesis of nucleic acids.

In some forms, a combination of PNAs, DNAs, and/or LNAs is used for themicrofluidic device-based synthesis of nucleic acids. In some forms, thenucleic acids produced according to the described methods are modifiedto incorporate fluorescent molecules. Exemplary fluorescent moleculesinclude fluorescent dyes and stains, such as Cy5 modified CTP.

b. Nucleotide Inhibitors

In some forms, component building blocks include nucleotide analogs thatinhibit or prevent addition of subsequent nucleotides to the growingnucleic acid, such as “inhibitory nucleotide analogs”. Exemplaryinhibitory nucleotide analogs include a charged inhibitory group that,upon incorporation into a growing nucleic acid, prevents subsequentnucleotide incorporation until the inhibitory group is removed.Therefore, in some forms, inhibitory nucleotide analogs include anucleotide triphosphate, a linker (or tether), a detectable label, and acharged inhibitory group, wherein the label and the inhibitory group areremovable.

In some forms, an inhibitor group can cause inhibition of subsequentnucleotide incorporation without steric hindrance. For example, theinhibition is caused by chemical or charge interaction with the enzymeand not be a physical blocking of the enzyme. In other forms, thecharged inhibitor also provides steric inhibition of enzyme activity.Therefore, in some forms, component building blocks include one or moreinhibitory nucleotide analogs including a charged inhibitor group thatprovides steric hindrance, or which does not provides steric hindrance.

In some forms, the inhibitor moiety is negatively charged or capable ofbecoming a negatively charged. In other forms, the inhibitor moiety ispositively charged or capable of becoming positively charged. In someforms, the Inhibitor includes a charged moiety (e.g., a negativelycharged moiety, a positively charged moiety, or both) or a moiety thatis capable of becoming charged. The Inhibitor can include two or morecharged groups. In some forms, the Inhibitor includes a charged groupselected from the group consisting of —COOH, —PO4, —SO4, —SO3, —SO2,—NRwRv, where Rw and Rv independently is H, an alkyl or aryl group. Insome forms, the inhibitor moiety does not comprise a —PO4 group. In someother forms, the inhibitor moiety does not comprise an aryl group. Incertain other forms, the inhibitor does not include a nucleotide ornucleoside or analogs thereof.

2. Amino Acid Component Building Blocks

In some forms, the component building blocks are naturally occurringamino acids, or derivatives thereof. For example, when a polypeptidesequence is synthesized, the microfluidic device (e.g., EWOD chip) isloaded with one or more reservoirs including one or more amino acids ina suitable buffer. Exemplary buffers include sterile filtered water andphysiological saline.

Exemplary amino acid component building blocks include, but are notlimited to the twenty standard amino acids (alanine, glycine, cysteine,arginine, aspartic acid, asparagine, histidine, lysine, glutamine,methionine, glutamic acid, threonine, proline, leucine, serine, valine,isoleucine, phenylalanine, tyrosine, tryptophan) in L-forms or D-forms,and modified variants thereof.

a. Modified Amino Acids

In some forms, the amino acid component building blocks are modifiedamino acids. For example, any of the twenty standard amino acids ca bemodified by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. Additional modifications include acetylation,propionylation, methylation, myristoylation, palmitoylation to add oneor more acetyl, methyl, myristoyl, or palmitoyl groups to an amino acid.Exemplary modified amino acids include hydroxy proline,γ-carboxyglutamate, O-phosphoserine, O-alanine, α-amino butyric acid,γ-amino butyric acid, α-amino isobutyric acid, ε-amino caproic acid,7-amino heptanoic acid, β-aspartic acid, ε-glutamic acid, cysteine(ACM), ε-lysine, ε-lysine (A-Fmoc), methionine sulfone, norleucine,norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxyproline, and thioproline.

b. Amino Acid Inhibitors

In some forms, component building blocks include amino acid analogs thatinhibit or prevent addition of subsequent amino acids to the growingpolypeptide, such as “inhibitory amino acid analogs”. Exemplaryinhibitory amino acid analogs include a charged inhibitory group that,upon incorporation into a growing polypeptide, prevents subsequent aminoacid incorporation until the inhibitory group is removed. Therefore, insome forms, inhibitory amino acids include a linker (or tether), adetectable label, and a charged inhibitory group, wherein the label andthe inhibitory group are removable.

In some forms, component building blocks include a peptide of 2 to 20units of amino acids or analogs, a peptide of 2 to 10 units of aminoacids or analogs, a peptide of 3 to 7 units of amino acids or analogs, apeptide of 3 to 5 units of amino acids or analogs. In some embodiments,the Inhibitor includes a group selected from the group consisting ofGlu, Asp, Arg, His, and Lys, and a combination thereof (e.g., Arg,Arg-Arg, Asp, Asp-Asp, Asp, Glu, Glu-Glu, Asp-Glu-Asp, Asp-Asp-Glu orAspAspAspAsp). Peptides or groups may be combinations of the same ordifferent amino acids or analogs.

3. Carbohydrate Component Building Blocks

In some forms, the component building blocks are naturally occurringmonosaccharides, or derivatives thereof. For example, when aoligosaccharide sequence is synthesized, the microfluidic device (e.g.,EWOD chip) is loaded with one or more reservoirs including one or moremonosaccharides in a suitable buffer. Exemplary buffers include sterilefiltered water and physiological saline.

Exemplary monosaccharide component building blocks include, but are notlimited to glucose (dextrose), fructose, galactose, ribose, xylose,allose, N- or O-substituted derivatives of neuraminic acid, and modifiedvariants thereof. In some forms, the monosaccharide component buildingblocks can be α-anomers, or β-anomers of D-isomers, L-isomers, orcombinations thereof.

In some forms, monosaccharide component building blocks are modifiedwith lipids,

4. Other Polymer Building Blocks

For example, a non-limiting list of polymer building blocks that can becoupled to synthetic nucleic acids prepared using microfluidicdevice-based methods includes poly(beta-amino esters); aliphaticpolyesters; polyphosphoesters; poly(L-lysine) containing disulfidelinkages; SOMAMERS® (see, Hensley, Journal of Biomolecular Techniques:TBT. 2013; 24(Suppl):S5); poly(ethylenimine) (PEI); disulfide-containingpolymers such as DTSP or DTBP crosslinked PEI; PEGylated PEI crosslinkedwith DTSP; Crosslinked PEI with DSP; Linear SS-PEI; DTSP-Crosslinkedlinear PEI; branched poly(ethylenimine sulfide) (b-PEIS). Typically, thepolymer has a molecular weight of between 500 Da and 20,000 Da,inclusive, for example, approximately 1,000 Da to 10,000 Da, inclusive.In some forms, the polymer is ethylene glycol. In some forms, thepolymer is polyethylene glycol. In an exemplary form, one or morepolymer are conjugated to the modified nucleic acids at one or morepositions in the sequence.

F. Enzyme Catalysts

Methods for template-free synthesis of biopolymers require catalysts toenable the addition of each component building block onto the initiatorsequence. Useful catalysts enable or increase the rate of incorporationof a component building block onto the biopolymer.

Exemplary catalysts enzymes are matched to a corresponding initiatorsequence. For example, in some forms, the initiator sequence is selectedaccording to class and composition of the catalyst used for thesynthesis.

In some forms, the catalyst includes one or more sequences designed tohybridize or otherwise bind to a solid support or stationary-phaseobjects such as magnetic beads, surfaces, agarose or other polymerbeads. In other instances, the catalyst includes one or more sites forconjugation to a molecule. For example, the catalyst can be conjugatedto a protein, or non-protein molecule, for example, to enableaffinity-binding of the catalyst, for example, to remove the catalystfrom the synthesized polymer.

1. Enzyme Catalysts for Nucleic Acid Synthesis

Exemplary catalysts useful for the enzymic template-free synthesis ofnucleic acids include Terminal deoxynucleotidyl transferases (TdT),Telomerases and Qbeta replicases.

a. Terminal Deoxynucleotidyl Transferases

Terminal deoxynucleotidyl transferase (TdT), also known as DNAnucleotidylexotransferase (DNTT), or terminal transferase, is aspecialized DNA polymerase.

TdT is a template independent polymerase that catalyzes the addition ofdeoxynucleotides to the 3′ hydroxyl terminus of DNA molecules. TdT is amember of the Pol X family TdT catalyses the template-free addition ofnucleotides to the 3′ terminus of a DNA molecule. The preferredsubstrate of this enzyme is a 3′-overhang, but it can also addnucleotides to blunt or recessed 3′ ends. Cobalt is a necessarycofactor, however the enzyme catalyzes reaction upon Mg and Mnadministration in vitro. TdT does not discriminate among the four basepairs when adding them to the N-nucleotide segments, it has shown a biasfor guanine and cytosine base pairs.

TdT is used to add labeled nucleotides to one or more termini of anucleic acid (e.g., DNA). for radio-labeling, cloning, and otherlabeling strategies. Commercially sources of TdT enzymes are known inthe art (e.g., NEB Catalog. #M0315).

In some forms, the DNA polymerase is DNA polymerase mu (Pol μ). Pol μdisplays intrinsic terminal deoxynucleotidyltransferase activity and astrong preference for activating Mn²⁺ ions.

A number of error-prone DNA polymerases efficiently incorporatenucleotides in DNA lesions where template information is missing(Goodman, Annu Rev Biochem. 71:17-50 (2002)). In some forms, the DNApolymerase is a Y-family DNA polymerase. Rev1, which was originallyidentified and isolated because of its UV-induced expression and UVsensitivity in its absence, is present universally among eukaryotes.Rev1 is a template-independent deoxycytidyl transferase (Lawrence C W etal., J. Mol. Biol. 122(1), 1-21(1978)). Protruding, recessed orblunt-ended double or single-stranded DNA molecules serve as a substratefor TdT. The 58.3 kDa enzyme does not have 5′ or 3′ exonucleaseactivity. The addition of Co′ in the reaction makes tailing moreefficient.

An exemplary reaction buffer for TdT includes 50 mM Potassium Acetate,20 mM Tris-acetate, and 10 mM Magnesium Acetate (pH 7.9 @ 25° C.)

b. Telomerase

Telomerase is another example of a DNA-template free polymerase.Telomerase is a special reverse transcriptase that extends one strand ofthe telomere repeat by using a template embedded in an RNA subunit.However, in the presence of manganese, both yeast and human telomerasecan switch to a template- and RNA-independent mode of DNA synthesis,acting in effect as a terminal transferase (Lue, et al., PNAS. 102 (28)9778-9783 (2005)).

c. Q-Beta Replicase

Qbeta replicase is another example of template free polymerase fornucleic acids, in particular for RNA (Biebricher et al., Nature.321(6065):89-91(1986) Biebricher et al., EMBO J, 15(13): 3458-3465(1996)).

RNA-dependent RNA polymerase (RdRP), (RDR), or RNA replicase, is anenzyme that catalyzes the replication of RNA from an RNA template. Thisis in contrast to a typical DNA-dependent RNA polymerase, whichcatalyzes the transcription of RNA from a DNA template.

G. Buffers and Wash Reagents

In some forms, methods for microfluidic device-based synthesis ofbiopolymers employ buffers and wash reagents. Wash buffers can be anysolution that is used to remove or reduce the local concentration ofanother component, for example, an enzyme.

Exemplary buffers and wash reagents include water, physiological saltsolutions, for example, PBS, and DMEM.

1. Stop Reagents and Blocking Buffers

In some forms, methods for microfluidic device-based synthesis ofbiopolymers employ blocking buffers and stop reagents. Blocking buffersare used to prevent or reduce the activity of a catalyst, for example, apolymerase enzyme. In some forms, the stop or block reagent quenches theenzymic catalysis that incorporates the component building block ontothe growing biopolymer chain. Typically, the methods include stopreagents and/or blocking reagents that are specific or effective tostop, reduce or otherwise mediate the activity of the catalyst enzymethat is employed. Blocking buffers and stop reagents effective forspecific catalyst enzymes are known in the art.

In some forms, the methods include the enzyme TdT as a catalyst foraddition of nucleic acids to a nucleic acid biopolymer. Therefore, themethods provide inhibitors for the inhibition of TdT. Exemplaryinhibitors of TdT include metal chelators (e.g., EDTA), sodium,ammonium, chloride, iodide, phosphate ions, and TRIS buffer. Therefore,in some forms, the stop buffer for TdT includes one or more of EDTA,sodium, ammonium, chloride, iodide, phosphate ions, and TRIS buffer.Exemplary inhibitors of TdT polymerase include Genistin and Heptelidicacid.

Exemplary inhibitors of telomerase enzymes include BIBR 1532, BRACO 19trihydrochloride, Costunolide, RHPS 4 methosulfate, TMPyP4 tosylate.Exemplary inhibitors of DNA polymerase include amikhelline, actinomycinD, aphidicolin, cytarabine, mithramycin A, 7-Aminoactinomycin D,rifamycin SV monosodium salt, 1-beta-D-Arabinofuranosylcytosine,2prime-O-Methyl Guanosine, acridine orange hemi(zinc chloride) salt,deacetylcolchiceine, Foscarnet sodium, rubrofusarin, rugulosin,resistomycin, juglone, alpha-amanitin, rifapentine, and vernolepin.Exemplary inhibitors of RNA polymerase include amatoxins (10 P), RNAPolymerase III Inhibitor, and rifamycin antibiotics, aureothricin,2prime-C-Methyl Cytidine, and Thiolutin.

In some forms, stop reagents include one or more inhibitory componentbuilding blocks, for example, one or more inhibitory nucleotide analogs,or one or more inhibitory amino acids.

In some forms, stop reagents include molecules that immediately preventactivity of a catalyst enzyme. An exemplary agent that immediatelyprevents the activity of a catalyst enzyme is a molecule that sequestersand/or chelates one or more enzyme co-factors. Exemplary co-factor thatcan be sequestered include ions, such as metal ions.

In some forms, a stop reagent includes one or more molecules thatchelate ions. In some forms, the methods include chelating agents thatchelate Mg2+ ions. Chelating agents that chelate enzyme co-factors areknown in the art. Exemplary chelating agents include EDTA, BAPTA andEGTA.

EDTA (ethylenediaminetetraacetic acid) is an aminopolycarboxylic acidand a colorless, water-soluble solid. Its conjugate base isethylenediaminetetraacetate. It is a widely used chelating agent tosequester metal ions such as Ca2+ and Fe3+. After being bound by EDTAinto a metal complex, metal ions remain in solution but exhibitdiminished reactivity. EDTA is produced as several salts, notablydisodium EDTA and calcium disodium EDTA.

EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraaceticacid), also known as egtazic acid (INN, USAN), is an aminopolycarboxylicacid, a chelating agent. It is a colourless solid that is related to thebetter known EDTA. Compared to EDTA, it has a lower affinity formagnesium, making it more selective for calcium ions.

In some forms, the activity of one or more stop or blocking reagents isenhanced or enabled by one or more external factors. For example, insome forms, TdT enzymes are inactivated by heating at 70° C. for 10minutes. The heating can occur in the presence of one or more stopreagents, such as EDTA.

H. Encapsulation Agents

In some forms, sequence-encoded polymers are packaged into discrete SMOsvia encapsulation. Suitable encapsulating agents include gel-basedbeads, protein viral packages, micelles, mineralized structures,siliconized structures, or polymer packaging.

In some forms, the encapsulating agents are viral capsids or afunctional part, derivative and/or analogue thereof. In some forms, theencapsulating agents are lipids forming micelles, or liposomessurrounding the nucleic acid encoding a format of information. In someforms, the encapsulating agents are natural or synthetic polymers. Insome forms, the encapsulating agents are mineralized, for example,calcium phosphate mineralization of alginate beads, or polysaccharides.In other forms, the encapsulating agents are siliconized. Packaging ofbitstream polymer sequences into memory blocks allows for selection andsuperstructuring by use of molecular identifiers, or “addresses”. Inaddition to nucleic acid overhangs, other purification tags can beincorporated into the overhang nucleic acid sequence in any SMOs forpurification (i.e. data retrieval). In some forms, the overhang containsone or more purification tags. In some forms, the overhang containspurification tags for affinity purification. In some forms, the overhangcontains one or more sites for conjugation to a nucleic acid, ornon-nucleic acid molecule. For example, the overhang tag can beconjugated to a protein, or non-protein molecule, for example, to enableaffinity-binding of the SMOs. Exemplary proteins for conjugating tooverhang tags include biotin, antibodies, or antigen-binding fragmentsof antibodies.

I. Reagents for Modification of Biopolymers

Biopolymers designed and synthesized according to the describedmicrofluidic device-based methods can be modified to add, remove, modifyor otherwise interact with molecules having a known function.

Exemplary modifying moieties can be selected according to thebiopolymer, and can include small molecules, proteins, peptides, nucleicacids, lipids, saccharides, or polysaccharides.

a. Enzymes for Modifying Nucleic Acids

Enzymes that modify one or more components of a nucleic acid biopolymerare described for use with the described methods. Enzymes that degrade,cleave or otherwise remove one or more nucleotides at one or more siteswithin a nucleic acid are provided.

i. Exonucleases

In some forms the methods employ one or more exonucleases to remove oneor more nucleic acids from either end of a nucleic acid biopolymer.Exonuclease enzymes, and appropriate buffer conditions for optimalexonuclease activity are known in the art. Exemplary exonuclease enzymesinclude Lambda Exonuclease, E. coli Exonuclease I, Exonuclease II, E.coli Exonuclease III, Exonuclease V, Exonuclease VI, Exonuclease VII,and Exonuclease T.

ii. Endonucleases

In some forms the methods employ one or more endonucleases to remove oneor more nucleic acids from within a nucleic acid biopolymer.Endonuclease enzymes, and appropriate buffer conditions for optimalexonuclease activity are known in the art. Exemplary endonucleaseenzymes include Mung Bean Nuclease, DNase I, Micrococcal Nuclease, T7Endonuclease I, Thermostable FEN1, and Nuclease BAL-31.

iii. Restriction Endonucleases

In some forms the methods employ one or more restriction endonucleasesto cut, cleave or remove one or more nucleic acids at asequence-controlled region of a biopolymer. Restriction endonucleases(RE) are enzymes that cut the sugar-phosphate backbones of complementarynucleic acids within the DNA double helix to produce blunt-ended nucleicacid fragments (i.e., both strands terminate in a base pair).Restriction endonuclease enzymes that recognize a specific sequence ofnucleotides and cut both strands of DNA to yield blunt-ended DNAfragments are well known in the art. Recognition sequences forrestriction endonuclease enzymes are generally between 4 and 8 bases.Restriction endonuclease enzymes that digest double stranded DNA toproduce a blunt-ended DNA fragments (i.e., blunt-cutting RE) canrecognize palindromic or non-palindromic sequences. The cut site can bewithin the recognition sequence, or can be contiguous with therecognition sequence, or at a distance from the recognition sequence. Anon-limiting list of blunt-end restriction endonuclease enzymes includesAanI, Acc16I, AccBSI, AccII, AcvI, AfaI, AfeI, AhaIII, AjiI, AleI,AluBI, AluI, Aor51HI, Asp700I, AssI, BalI, BbrPI, BmcAI, BmgBI, BmiI,BoxI, BsaAI, BsaBI, Bse8I, BseJI, Bsh1236I, BshFI, BsnI, Bsp68I, BspFNI,BspLI, BsrBI, BssNAI, Bst1107I, BstBAI, BstC8I, BstFNI, BstPAI, BstSNI,BstUI, BstZ17I, BsuRI, BtrI, BtuMI, Cac8I, CdiI, CviJI, CviKI_1, CviRI,DinI, DpnI, DraI, Ec113611, Eco105I, Eco147I, Eco32I, Eco47III, Eco53kI,Eco72I, EcoICRI, EcoRV, EgeI, EheI, EsaBC3I, FaiI, FnuDII, FspAI, FspI,GlaI, Had, HaeIII, HincII, HindII, HpaI, Hpyl66II, Hpy8I, HpyCH4V,KspAI, LpnI, MalI, MbiI, MlsI, MluNI, MlyI, MroXI, MscI, Ms1I, Msp20I,MspA1I, MssI, MstI, MvnI, NaeI, NlaIV, NruI, NsbI, NspBII, OliI, PceI,PdiI, PdmI, PmaCI, PmeI, PmlI, Ppu21I, PshAI, PsiI, PspCI, PspN4I,PvuII, RruI, RsaI, RseI, ScaI, SchI, SciI, SfoI, SmaI, SmiI, SmiMI,SnaBI, SrfI, SseBI, SspD5I, SspI, Sth302II, StuI, SwaI, XmnI, ZraI, andZrmI

V. Uses

The described methods and compositions for automated template-freesynthesis and manipulation of sequence controlled biopolymers can beused for a wide range of applications. Exemplary applications includepreparation and organization of biopolymer-based memory systems.

A. Microfluidic Synthesis for Nucleic Acid Memory

The described methods for the design, synthesis and/or manipulation ofbiopolymers using microfluidic devices can be implemented for automatedlarge-scale simultaneous production of a multiplicity of uniquelyaddressed, user-defined biopolymers.

The methods can synthesize biopolymers for use in a wide variety ofapplications, including for biopolymer-based memory storage. In someforms, the methods include organizing information within memory storageunits, such as nucleic acid, or polypeptide encapsulation units, throughmovement of droplets actuated through a microfluidics platform. Infurther forms, the methods include retrieving the bitstream-encodedsequence from the biopolymer memory storage units.

1. Nucleic Acid Memory Storage

Methods of synthesizing and manipulating user-defined nucleic acids formemory storage are provided. In some forms, microfluidic systmes areimplemented to synthesize and manipulate data-sequence nucleic acidsencoding a format of data are encapsulated within a layer of natural, orsynthetic material. A nucleic acid of any arbitrary form can beencapsulated, for example, a linear, a single-stranded, base-paireddouble stranded, or a scaffolded nucleic acid. Exemplary encapsulatingagents include proteins, lipids, saccharides, polysaccharides, nucleicacids, and any derivatives thereof, as well as hydrogel and syntheticpolymers including polystyrene, or silica, glass, and paramagneticmaterials. These encapsulated nucleic acids form discrete memory storageunits that allow for controlled segregation of blocks of information. Insome forms, the methods also optionally include organizing informationwithin nucleic acid memory storage units. In some forms, the methodsalso optionally include accessing the data-encoded sequence, forexample, accessing bitstream-encoded data from an enclosed nucleic acidsequence. In some forms, the methods also include steps of retrievingthe bitstream-encoded sequence from the biopolymer memory storage units.

Methods for microfluidic-based production of biopolymers and particlesencapsulating biopolymers can be applied for the creation of nucleicacid memory objects for storage of information using nucleic acids ofany length, or any form have also been developed. Typically, nucleicacids of any desired length are packaged, encapsulated, enveloped, orencased in gel-based beads, protein viral packages, micelles,mineralized structures, siliconized structures, or polymer packaging,herein referred to as “nucleic acid package”. In some forms, linearnucleic acids, encoding a bitstream of information, are base-paired,double-stranded. In other forms, linear nucleic acids consist of a longcontinuous single-stranded nucleic acid polymer or many such polymers.These discrete nucleic acid packages serve as nucleic acid memoryobjects (NMOs) and allow incorporation of one or more specific tags onthe surface of the structures. Some exemplary tags include nucleic acidsequence tags, protein tags, carbohydrate tags, and any affinity tags.

The manner in which the indices/barcodes are attached to the externalsurface of the core particle and/or biopolymer sequence can be variedaccording to the desired manner for pooling, sorting, organizing andaccessing the information. In other forms, encapsulated particle areformed in which the “shell” that is the product of “shelling” containsthe encoded data.

Typically, the methods for assembling and storing a desired media assequence-controlled polymer memory object (SMO) include one or more ofthe following steps:

(A) Providing a bitstream encoded sequence containing the desired media;

(B) Creating a sequence-controlled polymer memory object (SMO) includingthe bitstream encoded sequence; and

(C) Storing the SMO including the bitstream encoded.

In some forms, the methods also include one or more of the followingsteps:

(D) Organizing or combining information within two or more SMOs;

(E) Retrieving the bit stream encoded sequence within one or moreselected SMOs; and

(F) Accessing the media encoded within the selected SMO.

Each of these steps can be implemented within microfluidic devices tocontrol the movement of droplets or fluid flow to organize thesynthesis, manipulation, storage and retrieval of encoded information.

a. Conversion of Data to Biopolymer Sequence

Typically, the methods require providing a polymer sequence that encodesa piece of desired information, such as bitstream data. Suitablepolymers include sequence-controlled polymers, such as macromoleculescomposed of a non-random sequence of discrete monomers. An exemplarysequence-controlled polymer is a nucleic acid, such as single ordouble-stranded DNA, or RNA. For example, in some forms, asingle-stranded nucleic acid sequence encoding bitstream data is inputfor the design of a nucleic acid nanostructure having a user-definedshape and size.

In some forms, a portion or portions of a digital format of information,such as an html format of information or any other digital format suchas a book with text and/or images, audio, or movie data, is converted tobits, i.e., zeros and ones. In some forms, the information can beotherwise converted from one format (e.g., text) to other formats suchas through compression by Lempel-Ziz-Markov chain algorithm (LZMA) orother methods of compression, or through encryption such as by AdvancedEncryption Standard (AES) or other methods of encryption. Other formatsof information that can be converted to bits are known to those of skillin the art.

Therefore, in some forms, the methods include converting a format ofinformation into one or more bit sequences of a bit stream. One or morebit sequences can be converted into one or more corresponding polymersubunits. In an exemplary form, bit sequences are converted to nucleicacid sequences. Methods for converting bit sequences into one or moresequence-controlled polymers are known in the art.

In exemplary forms, a digital file, encoded on a computer as a bitstream of 0's and 1's, is reversibly converted to a nucleic acidsequence using any of the methods known in the art.). In some forms, anoligonucleotide or DNA using a 1 bit per base encoding (A or C=0; T orG=1) to form a corresponding encoded oligonucleotide sequence, i.e. theoligonucleotide sequence corresponds to or encodes for the bit sequence.In some forms the choice of digital format, for example the encryptionsalt, and the choice of bitstream to equivalent nucleic acid sequence,for example choice of A rather than C, is optimized such that thesequence repetition and sequence self-complementarity are avoided,identified by methods known to the art.

The nucleic acid sequence generated from the bit stream data of adesired media is termed the “bit stream encoded sequence”. The bitstream data encoded within the long scaffold sequence is typically“broken-up” into fragments. For example, data can be fragmented into anysize range from about 100 to about 1,000,000 nucleotides, such as fromabout 375 to about 51,000 bases, inclusive, per object, for example, 500bp up to 50,000 bp. In the digital storage field this is conceptuallysynonymous with “page” or “block”. The bit stream-encoded nucleic acidsequence is synthesized according to the described template-freesynthesis methods using a microfluidic device, and is optionallyamplified or purified using a variety of known techniques (i.e.,asymmetric PCR, bead-based purification and separation, cloning andpurification).

In some forms, the memory page will have identifying information as partof each sequence, including a file format signature, a sequence encodingan encryption salt, a unique identifying page number, a memory blocklength, and a sequence for DNA amplification.

In an exemplary form, a digital file is compressed, for example, usingthe LZMA method, or the file is encrypted, for example, using AES128encryption using a supplied password.

In some forms, the methods include syntesizing, or otherwose providing anucleic acid sequence from a pool containing a multiplicity of similaror different sequences. In some forms, the pool is a database of knownsequences. For example, in certain forms a discrete “block” ofinformation is contained within a pool of nucleic acid sequences rangingfrom about 100-1,000,000 bases in size, though this upper limit istheoretically unlimited. In some forms, the nucleic acid sequenceswithin a pool of multiple nucleic acid sequences share one or morecommon sequences. When nucleic acids that are provided are selected froma pool of sequences, the selection process can be carried out manually,for example, by selection based on user-preference, or automatically.

b. Assembly of Memory Objects

Assembly of memory objects by encapsulation, or direct assembly ofsequence-encoded biopolymers and address tags/barcodes can be carriedout according the described microfluidic-based methods to produce memoryobjects having a range of different structures. For example, in someforms, memory objects include a core particle, onto which one or moresequence-encoded biopolymers is bound. Binding of sequence encodedbiopolymers to a particle core can be achieved according to themicrofluidic methods, for example, using enzymes to caltalyze covalentor non-covalent linkages. In some forms, a core molecule is coated orcoupled to a molecule which is an intermediary receptor, for example, abinding site that is recognized by one or more ligands associated withthe sequence encoded biopolymer.

In some forms, sequence-encoded biopolymers are coupled or hybridized toa receptor-coated core molecule. In some forms, the polymer/coresubstructure is then coated with one or more encapsulating agents (i.e.,“molecular shelling”) to produce a coated polymer/core structure, whichis then coupled to one or more address labels, or barcodes.

Binding of address labels to a coated polymer/core particle can beachieved using covalent or non-covalent linkages, or hybridization ofcomplementary nucleic acids. In some forms, assembly of a memory objectincludes loading or complexing one or more sequence-encoded biopolymerswithin the interior space(s) of a porous, or otherwise accessiblepolymer core molecule or structure. In some forms, assembly of a memoryobject includes encapsulating, or shelling the polymer-loaded core tocreate an encapsulated polymer-loaded particle, which is then complexedwith one or more address tags or barcodes.

In some forms, memory objects include a sequence-encoded polymer, andoptionally core molecules and/or encapsulating agents that are coatedwith multiple different types of address tags or barcodes. For example,in some forms, memory objects are assembled to enable multiplexedmolecular logic operations and data selection. For example, in someforms, encapsulation or molecular shelling of one or moresequence-encoded biopolymers, including multiple pieces of bit-streamencoded data are labelled with multiple address tags or barcodes. Theaddress tags or barcodes can be attached directly to the molecular core,or absorbed by a molecular core are further surrounded by a molecularshell and functionalized with addressing/specificity tags formultiplexed computation.

In some forms, the descibed methods for microfluidic-actuated movementof droplets synthesize biopolymers into memory objects including:

(i) one or more sequence-encoded biopolymers;

(ii) optionally core molecules or encapsulating agents that are coatedwith address tags or barcodes; and

(iii) a shell or core which itself produces a signal, or has anotherproperty that can be detected and measured to produce a readout.

The outer “shell”, or inner “core” of a memory particle can, therefore,be used to address or label the memory object. Exemplary physical orchemical properties that can be detected and measured include optical,magnetic, electric, or physical properties.

Therefore, in some forms, the outer shell or inner core of a memoryobject produces a readout based on optical, magnetic, electric, orphysical properties of the shell/core. Therefore, in some forms, datastreams are encoded directly on a molecular core, which has a readoutbased on optical, magnetic, electric, or physical properties of thecore. The molecular core also contains address/specificity tags formolecular logic and data retrieval operations. In some forms, the datastream is encoded on a molecular shell surrounding a molecular core. Theshell/core has readouts based on the optical, magnetic, electric, orphysical properties of the shell/core. The shell is functionalized withaddressing/specificity tags for molecular logic and data retrievaloperations.

Synthesized biopolymer memory objects prepared according to describedmicrofluidic methods are suitable for many applications. Some exemplaryuses include in memory storage, in nano-electronic circuitry, etc.Sequence-controlled biopolymer memory objects including nucleic acids orother sequence-controlled biopolymers that encode a format of data,encapsulated within natural, or synthetic material, are also provided.In some forms, a nucleic acid or other biopolymer of any arbitrary formcan be encapsulated. For example, in some forms a linear, asingle-stranded, a base-paired double stranded, or a scaffolded nucleicacid is encapsulated.

Exemplary encapsulating agents include proteins, lipids, saccharides,polysaccharides, nucleic acids, synthetic polymers, hydrogel polymers,silica, paramagnetic materials, and metals, as well as any derivativesthereof. These encapsulated nucleic acids or other biopolymer areassociated with one or more overhang nucleic acid sequences that areused for adding addresses, and/or purification tags. In some forms,multiple layers of encapsulation and overhang nucleic acids are designedfor additional sorting and tagging the format of information.

In some forms, the bit stream encoded nucleic acid sequence is not thesame sequence as chromosomal DNA, or mRNA, or prokaryotic DNA. Forexample, in some forms, the entire bit stream encoded sequence has lessthan 20% sequence identity to a naturally-occurring nucleic acidsequence, for example, less than 10% identity, or less than 5% identity,or less than 1% identity, up to 0.001% identity. In other forms, thebitstream sequences are composed of the sequences of cDNAs, genes,protein sequences, protein coding open reading frames, or biologicalsequences that together in a pool form a database of biologicalsequences.

The disclosed compositions and methods can be further understood throughthe following text.

In some forms, the method is a method for synthesis of a specificnucleic acid sequence programmed by the movement of nucleotides,enzymes, buffer, salts, and water in aqueous droplets usingelectrowetting on dielectric (EWOD) movement of droplets. In some forms,the method is a method of addressed location synthesis of nucleic acidpolymers by the movement of drops containing the next nucleic acid to beadded into the drop containing the growing synthesized polymer. In someforms, the microfluidic device is a chip design allowing for theaddition of nucleic acids in droplets on the EWOD chip in controlledvolumes for the addition to a growing polymer. In some forms, themicrofluidic device is a chip design for the stable fixation of agrowing nucleic acid polymer to a defined, addressed location on a chipused in EWOD droplet movement. In some forms, the method is a method ofsimultaneously carrying out instructions in parallel to massivelyparallelize the synthesis of many different sequences at many differentaddressed locations across the chip.

Disclosed are methods for synthesizing a biopolymer having a desiredsize and sequence in the absence of a template, where the methodcomprises: (a) combining, on a microfluidic device, a droplet comprisinga component initiation sequence with one or more droplets collectivelycomprising a component building block and an attachment catalyst to forma combined droplet; and (b) optionally repeating step (a) to perform thestep-wise addition of component building blocks to the biopolymer toform a biopolymer having a preselected, desired polymer sequence andlength. The droplets comprises a component initiation sequence and eachof the droplets collectively comprising the component building block andthe attachment catalyst were, prior to the combining, at differentlocations on the microfluidic device. One or more additional droplets,each comprising an additional component building block, are at differentlocations on the microfluidic device than the droplet comprising thecomponent sequence, the droplets collectively comprising the componentbuilding block and the attachment catalyst, or the combined droplet. Thecombining comprises conditions suitable for the attachment catalyst toattach the component initiation sequence to the component building blockto form a biopolymer.

In some forms, the conditions suitable for the attachment of thecomponent initiation sequence with the component building block to forma biopolymer in step (a) comprise contacting the combined droplet withone or more reagents selected from the group consisting of a washreagent, a blocking reagent, and a stop reagent. In some forms, each ofthe wash reagent, blocking reagent, and stop reagent are provided asindependent droplets on the microfluidic device. In some forms, thecombining of droplets in step (a) is accomplished by moving one or moreof the droplets on the microfluidic device using electrical chargeprovided by an optic fiber.

In some forms, the sequence of movement for each droplet on themicrofluidic device to produce the desired polymer sequence is providedin the form of a computer-readable program. In some forms, two or morebiopolymers are simultaneously or consecutively synthesized at differentlocations of the same microfluidic device. In some forms, the two ormore biopolymers have different sequences, different sizes, or bothdifferent sequences and different sizes. In some forms, each of the twoor more synthesized biopolymers is synthesized and purified at adistinct location on the same microfluidic device. In some forms, eachof the two or more biopolymers comprises a unique address tag.

In some forms, the component initiation sequence is coupled to a stablesupport matrix. In some forms, the support matrix is a bead. In someforms, the bead is magnetic.

In some forms, the droplet is an aqueous droplet having a volume betweenone femtoliter (fl) and 100 microliters (μl), preferably between onepicoliter (pl) and one nanoliter (nl). In some forms, the creation,movement and combination of the droplets on the microfluidic device iscontrolled by a computer program.

In some forms, the method further comprises (c) manipulating, purifying,or isolating the synthesized biopolymer on the microfluidic device. Insome forms, manipulating the synthesized biopolymer in step (c)comprises inducing one or more structural or functional changes in thebiopolymer. In some forms, isolating the synthesized biopolymer in step(c) comprises a complexity-reduction step. In some forms, thecomplexity-reduction step includes isolating the synthesized biopolymeron the basis of one or more properties selected from the groupconsisting of mass, size, electrochemical charge, hydrophobicity, pH,melting temperature, conformation, and affinity for one or more ligands.In some forms, manipulating the synthesized biopolymer in step (c)comprises incorporating into the biopolymer one or more labels selectedfrom the group consisting of a dye, a fluorescent molecule, aradiolabel, an affinity tag, and a barcode.

In some forms, the method further comprises, prior to step (a), formingone or more of the droplets comprising the component initiation sequenceand the droplets collectively comprising the component building blockand the attachment catalyst by splitting the droplets from reservoirsthat collectively comprise the component initiation sequence, thecomponent building block, and the attachment catalyst.

In some forms, the method further comprises, prior to step (a), formingone or more of the additional droplets by splitting the additionaldroplets from reservoirs that collectively comprise the additionalcomponent building blocks.

In some forms, the biopolymer is a nucleic acid. In some forms, thenucleic acid has a length of between 100 and 100,000 bases in length,between 200 and 10,000 bases in length, between 500 and 5,000 bases, orbetween 1,000 and 3,000 bases in length. In some forms, one or more ofthe component building blocks is selected from the group consisting ofadenosine, cytidine, guanosine, thymidine, uridine, inosine, uridine,xanthosine, and pseudouridine. In some forms, the nucleic acid issingle-stranded DNA.

In some forms, the attachment catalyst is a polymerase enzyme selectedfrom the group consisting of TdT, Qbeta replicase, and telomerase.

In some forms, step (c) comprises the polymerase chain reaction toamplify the synthesized nucleic acid.

In some forms, the method further comprises the step of sequencing thesynthesized nucleic acid.

In some forms, one or more droplets comprises a restriction endonucleaseand one or more suitable buffers for the effective function of therestriction endonuclease.

Also disclosed are methods for the automated manipulation of a nucleicacid sequence comprising combining, on a microfluidic device, thenucleic acid sequence and one or more endonuclease or exonucleaseenzymes, where the combining comprises conditions under which the one ormore endonuclease or exonuclease enzymes remove or degrade one or morenucleotides from the nucleic acid sequence to produce a degraded nucleicacid.

In some forms, the nucleic acid is immobilized on a solid support orsurface. In some forms, the method further comprises purifying thedegraded nucleic acid. In some forms, purifying the degraded nucleicacid comprises washing the degraded nucleic acid on the microfluidicdevice to remove the one or more endonuclease or exonuclease enzymes.

In some forms, the method further comprises adding one or morenucleotides to the degraded nucleic acid on the microfluidic device, toform a modified nucleic acid. In some forms, adding one or morenucleotides to the degraded nucleic acid comprises: (a) combining, onthe microfluidic device, a droplet comprising the degraded nucleic acidwith one or more droplets collectively comprising a component buildingblock and an attachment catalyst to form a combined droplet; and (b)optionally repeating step (a) one or more times. The droplets comprisethe degraded nucleic acid and each of the droplets collectivelycomprising the component building block and the attachment catalystwere, prior to the combining, at different locations on the microfluidicdevice. The combining comprises conditions suitable for the attachmentcatalyst to attach the degraded nucleic to the component building blockto form a modified nucleic acid.

In some forms, the nucleic acid is encodes bitstream data. In someforms, the manipulation is carried out in a region of the nucleic acidthat is a barcode. In some forms, the microfluidic device is anelectrowetting on dielectric (EWOD) device. In some forms, the nucleicacid is a barcode. In some forms, the barcode is attached to a nucleicacid memory object. In some forms, the barcode is not the exact sequenceof the barcode associated to the concept or metadata, but it mutatedaway from the barcode by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 mutations.

In some forms, the mutated barcode is associated with metadata or aconcept of the nearest barcode held in a barcode hash table associatingto metadata contained within the nucleic acid memory object. In someforms, the mutated barcode is associated with variations of metadata ora concept of the nearest barcode held in a barcode hash table. In someforms, the barcode is associated with metadata describing biologicalinformation of the nucleic acid sequence contained in the nucleic acidmemory object. In some forms, the nucleic acid sequence is encapsulatedwithin a nucleic acid memory object, where the nucleic acid memoryobject encodes a gene, and the barcode sequence describes one or morefeatures selected from the group consisting of gene name, mutations ofthe gene, the source organism, gene length, the protein(s) encoded thegene, and one or more ligands of the encoded protein.

In some forms, the barcode is associated with metadata describing thedigital information contained in a DNA sequence contained in the nucleicacid memory object. In some forms, the nucleic acid sequence encodesinformation about an image or images, and the metadata barcode containsthe amount of any given characteristic in the image, and where one ormore point mutations of the barcode of are associated with variedamounts of that characteristic. In some forms, the characteristic of theimage is the intensity of one or more colors. In some forms, the DNAsequence encodes a digital representation of an image or images, and themetadata barcode contains descriptions of objects in the image orimages, where the mutations of the barcodes of claim 42 are associatedwith the likeness to the object.

The disclosed compositions and methods can be further understood throughthe following numbered paragraphs.

1. A method for synthesizing a biopolymer having a desired size andsequence in the absence of a template, the method comprising:

(a) combining, on a microfluidic device, a droplet comprising acomponent initiation sequence with one or more droplets collectivelycomprising a component building block and an attachment catalyst to forma combined droplet,

wherein the droplets comprising a component initiation sequence and eachof the droplets collectively comprising the component building block andthe attachment catalyst were, prior to the combining, at differentlocations on the microfluidic device,

wherein one or more additional droplets, each comprising an additionalcomponent building block, are at different locations on the microfluidicdevice than the droplet comprising the component sequence, the dropletscollectively comprising the component building block and the attachmentcatalyst, or the combined droplet,

wherein the combining comprises conditions suitable for the attachmentcatalyst to attach the component initiation sequence to the componentbuilding block to form a biopolymer; and

(b) optionally repeating step (a) to perform the step-wise addition ofcomponent building blocks to the biopolymer to form a biopolymer havinga preselected, desired polymer sequence and length.

2. The method of paragraph 1, wherein the conditions suitable for theattachment of the component initiation sequence with the componentbuilding block to form a biopolymer in step (a) comprise contacting thecombined droplet with one or more reagents selected from the groupconsisting of a wash reagent, a blocking reagent, and a stop reagent.3. The method of paragraph 2, wherein each of the wash reagent, blockingreagent, and stop reagent are provided as independent droplets on themicrofluidic device.4. The method of paragraph 1, wherein the combining of droplets in step(a) is accomplished by moving one or more of the droplets on themicrofluidic device using electrical charge provided by an optic fiber.5. The method of any one of paragraphs 1-4, wherein the sequence ofmovement for each droplet on the microfluidic device to produce thedesired polymer sequence is provided in the form of a computer-readableprogram.6. The method of paragraph 1, wherein two or more biopolymers aresimultaneously or consecutively synthesized at different locations ofthe same microfluidic device.7. The method of paragraph 6, wherein the two or more biopolymers havedifferent sequences, different sizes, or both different sequences anddifferent sizes.8. The method of paragraph 7, wherein each of the two or moresynthesized biopolymers is synthesized and purified at a distinctlocation on the same microfluidic device.9. The method of paragraph 8, wherein each of the two or morebiopolymers comprises a unique address tag.10. The method of any one of paragraphs 1-9, wherein the componentinitiation sequence is coupled to a stable support matrix.11. The method of paragraph 10, wherein the support matrix is a bead.12. The method of paragraph 11, wherein the bead is magnetic.13. The method of any one of paragraphs 1-12, wherein the droplet is anaqueous droplet having a volume between one femtoliter (fl) and 100microliters (μl), preferably between one picoliter (pl) and onenanoliter (nl).14. The method of any one of paragraphs 1-13, wherein the creation,movement and combination of the droplets on the microfluidic device iscontrolled by a computer program.15. The method of any one of paragraphs 1-14, further comprising

(c) manipulating, purifying, or isolating the synthesized biopolymer onthe microfluidic device.

16. The method of paragraph 15, wherein manipulating the synthesizedbiopolymer in step (c) comprises inducing one or more structural orfunctional changes in the biopolymer.17. The method of paragraph 16, wherein isolating the synthesizedbiopolymer in step (c) comprises a complexity-reduction step.18. The method of paragraph 17, wherein the complexity-reduction stepincludes isolating the synthesized biopolymer on the basis of one ormore properties selected from the group consisting of mass, size,electrochemical charge, hydrophobicity, pH, melting temperature,conformation, and affinity for one or more ligands.19. The method of paragraph 16, wherein manipulating the synthesizedbiopolymer in step (c) comprises incorporating into the biopolymer oneor more labels selected from the group consisting of a dye, afluorescent molecule, a radiolabel, an affinity tag, and a barcode.20. The method of any one of paragraphs 1-19 further comprising, priorto step (a), forming one or more of the droplets comprising thecomponent initiation sequence and the droplets collectively comprisingthe component building block and the attachment catalyst by splittingthe droplets from reservoirs that collectively comprise the componentinitiation sequence, the component building block, and the attachmentcatalyst.21. The method of paragraph 20 further comprising, prior to step (a),forming one or more of the additional droplets by splitting theadditional droplets from reservoirs that collectively comprise theadditional component building blocks.22. The method of any one of paragraphs 1-21, wherein the biopolymer isa nucleic acid.23. The method of paragraph 22, wherein the nucleic acid has a length ofbetween 100 and 100,000 bases in length, between 200 and 10,000 bases inlength, between 500 and 5,000 bases, or between 1,000 and 3,000 bases inlength.24. The method of paragraph 22 or 23, wherein one or more of thecomponent building blocks is selected from the group consisting ofadenosine, cytidine, guanosine, thymidine, uridine, inosine, uridine,xanthosine, and pseudouridine.25. The method of paragraph 23, wherein the nucleic acid issingle-stranded DNA.26. The method of any one of paragraphs 22-25, wherein the attachmentcatalyst is a polymerase enzyme selected from the group consisting ofTdT, Qbeta replicase, and telomerase.27. The method of any one of paragraphs 22-26, wherein step (c)comprises the polymerase chain reaction to amplify the synthesizednucleic acid.28. The method of any one of paragraphs 22-27, further comprising thestep of sequencing the synthesized nucleic acid.29. The method of any one of paragraphs 22-27, wherein one or moredroplets comprises a restriction endonuclease and one or more suitablebuffers for the effective function of the restriction endonuclease.30. A method for the automated manipulation of a nucleic acid sequencecomprising combining, on a microfluidic device, the nucleic acidsequence and one or more endonuclease or exonuclease enzymes,

wherein the combining comprises conditions under which the one or moreendonuclease or exonuclease enzymes remove or degrade one or morenucleotides from the nucleic acid sequence to produce a degraded nucleicacid.

31. The method of paragraph 30, wherein the nucleic acid is immobilizedon a solid support or surface.32. The method of paragraph 30 or 31, further comprising purifying thedegraded nucleic acid.33. The method of paragraph 32, wherein purifying the degraded nucleicacid comprises washing the degraded nucleic acid on the microfluidicdevice to remove the one or more endonuclease or exonuclease enzymes.34. The method of any one of paragraphs 30 to 33, further comprisingadding one or more nucleotides to the degraded nucleic acid on themicrofluidic device, to form a modified nucleic acid.35. The method of paragraph 34, wherein adding one or more nucleotidesto the degraded nucleic acid comprises:(a) combining, on the microfluidic device, a droplet comprising thedegraded nucleic acid with one or more droplets collectively comprisinga component building block and an attachment catalyst to form a combineddroplet,

wherein the droplets comprising the degraded nucleic acid and each ofthe droplets collectively comprising the component building block andthe attachment catalyst were, prior to the combining, at differentlocations on the microfluidic device,

wherein the combining comprises conditions suitable for the attachmentcatalyst to attach the degraded nucleic to the component building blockto form a modified nucleic acid; and

(b) optionally repeating step (a) one or more times.

36. The method of any one of paragraphs 30 to 35, wherein the nucleicacid is encodes bitstream data.37. The method of any one of paragraphs 30 to 36, wherein themanipulation is carried out in a region of the nucleic acid that is abarcode.38. The method of any one of paragraphs 1 to 37, wherein themicrofluidic device is an electrowetting on dielectric (EWOD) device.39. The method of any one of paragraphs 22 or 23, wherein the nucleicacid is a barcode.40. The method of paragraph 39, wherein the barcode is attached to anucleic acid memory object.41. The method of any one of paragraphs 39 or 40, wherein the barcode isnot the exact sequence of the barcode associated to the concept ormetadata, but it mutated away from the barcode by 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 mutations.42. The method of paragraph 41, wherein the mutated barcode isassociated with metadata or a concept of the nearest barcode held in abarcode hash table associating to metadata contained within the nucleicacid memory object.43. The method of paragraph 41, wherein the mutated barcode isassociated with variations of metadata or a concept of the nearestbarcode held in a barcode hash table.44. The method of any one of paragraphs 39-43, wherein the barcode isassociated with metadata describing biological information of thenucleic acid sequence contained in the nucleic acid memory object.45. The method of paragraph 44, wherein the nucleic acid sequence isencapsulated within a nucleic acid memory object, wherein the nucleicacid memory object encodes a gene, and the barcode sequence describesone or more features selected from the group consisting of gene name,mutations of the gene, the source organism, gene length, the protein(s)encoded the gene, and one or more ligands of the encoded protein.46. The method of any one of paragraphs 39-43, wherein the barcode isassociated with metadata describing the digital information contained ina DNA sequence contained in the nucleic acid memory object.47. The method of paragraph 46, wherein the nucleic acid sequenceencodes information about an image or images, and the metadata barcodecontains the amount of any given characteristic in the image, andwherein one or more point mutations of the barcode of are associatedwith varied amounts of that characteristic.48. The method of paragraph 47, wherein the characteristic of the imageis the intensity of one or more colors.49. The method of paragraph 46, wherein the DNA sequence encodes adigital representation of an image or images, and the metadata barcodecontains descriptions of objects in the image or images, wherein themutations of the barcodes of paragraph 42 are associated with thelikeness to the object.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Examples Example 1: Echo-Based Synthesis of Nucleic Acids in Solution

A destination 96-well plate was loaded with 3×16 wells containing 10 μMtdt polymerase from New England Biolabs in 1×tdt buffer supplied withthe reagent and an initiator sequence (GTCGTCGTCCCCTCAAACT) (SEQ ID NO:22) at 1 μM. 16 numbers were chosen for conversion to nucleotidesequences by using single-precision IEEE 754 binary code (pi, e,gravitational constant, Avagadro's number, Planck's constant, SIelectron volt, electron mass, proton mass, golden ratio, permittivity offree space, square root of 2, fine structure constant, hydrogenfrequency, Boltzmann constant, 1,000,000^(th) prime number, and a testsequence). The binary representation was then converted to nucleotidesequences by a Huffman coding scheme to allow for the data to be encodedin the nucleotide switch, such that A>T, T>C, and C>A homopolymerstretches were encoded 1, and A>C, T>A, and C>T homopolymer stretcheswere encoding for 0.

The sequences were then converted to a cherry pick list with nucleotidesbeing loaded into the source plate of an Echo 555 (LabCyte) anddistributed to the well that contains the sequence encoding the number,in triplicated. After each distribution for the wells, the destinationplate was removed and placed in a 37 C incubator for 15 minutes in highhumidity. Samples were removed after every 4 homopolymer stretches thatwere taken for gel analysis on a 10% polyacrylamide gel stained withSybrGold (ThermoFisher). The sequences were poly(A) tailed by additionof dATP as the final nucleotide. The second strand was completed by 4cycles with PCR with a poly(T) oligonucleotide primer, and size purifiedto enrich around 500 nucleotide length products.

The products were prepped for Illumina MiSeq 500×2 sequencing and thesequences were compiled to read out the encoded numbers.

Example 2: Developing Neighborhood Molecular Hash Barcodes

Two oligonucleotide primers were selected from a list of 240,000 knownorthogonal primers (Xu, et al., Proc Natl Acad Sci, 106 (7) 2289-2294(2009)). Pseudo-random mutations were generated for each of the primerssuch that the mutations were predicted to raise the binding energy byapproximately 20 kJ/mol, or approximately 5° C., with calculations madeby the ΔH and ΔS, when known.

Pseudo-random mutations that lowered too much or not enough wereremoved. Those primers that remained were mutated again with the samebinding energy constraint, until the list was pared down to an orderedlist of 11 primers with binding energies between adjacent primersdestabilized by 20 kJ/mol relative to binding energy between primers andtheir exact complements. These binding energies between abarcode-complement pair were chosen to be destabilized by an amountproportional to their distance from each other in the list of allpossible qualifying primers. Each of the two original primers producedan ordered list of 10 primer mutants, plus the original primer. Theseprimer neighborhoods were associated with two arbitrary metadata terms(“Red” and “Blue”) for description of images that are encoded in DNAsequences. The prescribed binding affinity relationship was verifiedexperimentally with a melting temperature assay. A 384-well plate wasgenerated with 10 mM Tris-HCl pH 8.1, 150 mM NaCl, 1 mM EDTA, and 2 μMper oligo of each possible primer-complement pair between “Red” primersand “Red” and “Blue” complements. 1×SybrGreen was added and aQuantStudio 6 was used to assay the melting temperature by imagingduring a temperature ramp (annealing from 95° C. to 25° C. and melting25° C. to 95° C., and repeating).

The melting temperature was calculated based on the inflection point ofthe melting curve, and these data plotted as a heat map. Perfect capturewas shown as a high melting temperature, while imperfect capture wasseen as a low melting temperature. Each temperature of melting wasassociated to the barcode pair in a matrix and a heatmap was generated.

The heatmap showed the expected results, with a high melting temperaturealong the diagonal of the red-like to red-like-complement strands, and afalling melting temperature with each successive mutation along bothaxes, while no specific binding was shown between the red barcodes andblue barcodes. For comparison, a computational heatmap was generated byusing the Santa-Lucia thermodynamic values, showing a high correlationwith the experimental results.

To validate the quantitative PCR melting experiment, UV/Vis monitoringabsorbance at 260 nm over the same temperature range was used todetermine the melting temperature. This was applied to the middle strand(50% “Red”-like barcode) against the other “Red”-like complementarystrands. The results of the melting experiment showed excellentagreement with the values from the quantitative PCR melting program.Thus, it was possible to predict “neighborhoods” of controlled sequencefor orthogonal barcoding with programmed noisy crosstalk.

Example 3: Barcode Selection by Fluorescent Bait Sequences

Fluorescent barcodes were purchased from IDT with sequencescomplementary to 3 barcodes chosen from the list of 240,000 orthogonalbarcodes (Xu, et al., Proc Natl Acad Sci, 106 (7) 2289-2294 (2009)),associated in an external table to be encoding “cat”, “wild”, and“orange”. 3 images of house cats (1 black and white, one brown, oneorange) and a tiger and a lion, and 2 house dogs (1 retriever, 1greyhound) and a wolf were encoded as 27×27 black and white images andconverted to DNA encoding after compression (run-length-encoding) andencryption of the bitmap image.

The DNA sequences were put into plasmid form and encapsulated in silicaas described above with methods in International Publication No. WO2017/189914.

The plasmids were barcoded with metadata tags such that approximately1,000 redundant barcode overhangs are present on each of the blocksencoding the images.

10× molar excess of the fluorescent strand was added to the barcodedmaterial and annealed at the predicted melting temperature. The unboundfraction was washed using 30 mM Tris HCl pH 8.1 and 150 mM NaCl inmultiple wash steps.

The barcoded images can be tested by fluorescence microscopy andfluorescent sorting, enabling rapid sorting using biochemical barcodingof plasmids and also digital information.

1. A method for synthesizing a biopolymer having a desired size andsequence in the absence of a template, the method comprising: (a)combining, on a microfluidic device, a droplet comprising a componentinitiation sequence with one or more droplets collectively comprising acomponent building block and an attachment catalyst to form a combineddroplet, wherein the droplets comprising a component initiation sequenceand each of the droplets collectively comprising the component buildingblock and the attachment catalyst were, prior to the combining, atdifferent locations on the microfluidic device, wherein one or moreadditional droplets, each comprising an additional component buildingblock, are at different locations on the microfluidic device than thedroplet comprising the component sequence, the droplets collectivelycomprising the component building block and the attachment catalyst, orthe combined droplet, wherein the combining comprises conditionssuitable for the attachment catalyst to attach the component initiationsequence to the component building block to form a biopolymer; and (b)optionally repeating step (a) to perform the step-wise addition ofcomponent building blocks to the biopolymer to form a biopolymer havinga preselected, desired polymer sequence and length.
 2. The method ofclaim 1, wherein the conditions suitable for the attachment of thecomponent initiation sequence with the component building block to forma biopolymer in step (a) comprise contacting the combined droplet withone or more reagents selected from the group consisting of a washreagent, a blocking reagent, and a stop reagent.
 3. The method of claim2, wherein each of the wash reagent, blocking reagent, and stop reagentare provided as independent droplets on the microfluidic device.
 4. Themethod of claim 1, wherein the combining of droplets in step (a) isaccomplished by moving one or more of the droplets on the microfluidicdevice using electrical charge provided by an optic fiber.
 5. The methodof claim 1, wherein the sequence of movement for each droplet on themicrofluidic device to produce the desired polymer sequence is providedin the form of a computer-readable program.
 6. The method of claim 1,wherein two or more biopolymers are simultaneously or consecutivelysynthesized at different locations of the same microfluidic device. 7.The method of claim 6, wherein the two or more biopolymers havedifferent sequences, different sizes, or both different sequences anddifferent sizes.
 8. The method of claim 7, wherein each of the two ormore synthesized biopolymers is synthesized and purified at a distinctlocation on the same microfluidic device.
 9. The method of claim 8,wherein each of the two or more biopolymers comprises a unique addresstag.
 10. The method of claim 1, wherein the component initiationsequence is coupled to a stable support matrix.
 11. The method of claim10, wherein the support matrix is a bead.
 12. The method of claim 11,wherein the bead is magnetic.
 13. The method of claim 1, wherein thedroplet is an aqueous droplet having a volume between one femtoliter(fl) and 100 microliters (μl), preferably between one picoliter (pl) andone nanoliter (nl).
 14. The method of claim 1, wherein the creation,movement and combination of the droplets on the microfluidic device iscontrolled by a computer program.
 15. The method of claim 1, furthercomprising (c) manipulating, purifying, or isolating the synthesizedbiopolymer on the microfluidic device.
 16. The method of claim 15,wherein manipulating the synthesized biopolymer in step (c) comprisesinducing one or more structural or functional changes in the biopolymer.17. The method of claim 16, wherein isolating the synthesized biopolymerin step (c) comprises a complexity-reduction step.
 18. The method ofclaim 17, wherein the complexity-reduction step includes isolating thesynthesized biopolymer on the basis of one or more properties selectedfrom the group consisting of mass, size, electrochemical charge,hydrophobicity, pH, melting temperature, conformation, and affinity forone or more ligands.
 19. The method of claim 16, wherein manipulatingthe synthesized biopolymer in step (c) comprises incorporating into thebiopolymer one or more labels selected from the group consisting of adye, a fluorescent molecule, a radiolabel, an affinity tag, and abarcode.
 20. The method of claim 1 further comprising, prior to step(a), forming one or more of the droplets comprising the componentinitiation sequence and the droplets collectively comprising thecomponent building block and the attachment catalyst by splitting thedroplets from reservoirs that collectively comprise the componentinitiation sequence, the component building block, and the attachmentcatalyst.
 21. The method of claim 20 further comprising, prior to step(a), forming one or more of the additional droplets by splitting theadditional droplets from reservoirs that collectively comprise theadditional component building blocks.
 22. The method of claim 1, whereinthe biopolymer is a nucleic acid.
 23. The method of claim 22, whereinthe nucleic acid has a length of between 100 and 100,000 bases inlength, between 200 and 10,000 bases in length, between 500 and 5,000bases, or between 1,000 and 3,000 bases in length.
 24. The method ofclaim 22, wherein one or more of the component building blocks isselected from the group consisting of adenosine, cytidine, guanosine,thymidine, uridine, inosine, uridine, xanthosine, and pseudouridine. 25.The method of claim 23, wherein the nucleic acid is single-stranded DNA.26. The method of claim 22, wherein the attachment catalyst is apolymerase enzyme selected from the group consisting of TdT, Qbetareplicase, and telomerase.
 27. The method of claim 22, wherein step (c)comprises the polymerase chain reaction to amplify the synthesizednucleic acid.
 28. The method of claim 22, further comprising the step ofsequencing the synthesized nucleic acid.
 29. The method of claim 22,wherein one or more droplets comprises a restriction endonuclease andone or more suitable buffers for the effective function of therestriction endonuclease.
 30. A method for the automated manipulation ofa nucleic acid sequence comprising combining, on a microfluidic device,the nucleic acid sequence and one or more endonuclease or exonucleaseenzymes, wherein the combining comprises conditions under which the oneor more endonuclease or exonuclease enzymes remove or degrade one ormore nucleotides from the nucleic acid sequence to produce a degradednucleic acid.
 31. The method of claim 30, wherein the nucleic acid isimmobilized on a solid support or surface.
 32. The method of claim 30,further comprising purifying the degraded nucleic acid.
 33. The methodof claim 32, wherein purifying the degraded nucleic acid compriseswashing the degraded nucleic acid on the microfluidic device to removethe one or more endonuclease or exonuclease enzymes.
 34. The method ofclaim 30, further comprising adding one or more nucleotides to thedegraded nucleic acid on the microfluidic device, to form a modifiednucleic acid.
 35. The method of claim 34, wherein adding one or morenucleotides to the degraded nucleic acid comprises: (a) combining, onthe microfluidic device, a droplet comprising the degraded nucleic acidwith one or more droplets collectively comprising a component buildingblock and an attachment catalyst to form a combined droplet, wherein thedroplets comprising the degraded nucleic acid and each of the dropletscollectively comprising the component building block and the attachmentcatalyst were, prior to the combining, at different locations on themicrofluidic device, wherein the combining comprises conditions suitablefor the attachment catalyst to attach the degraded nucleic to thecomponent building block to form a modified nucleic acid; and (b)optionally repeating step (a) one or more times.
 36. The method of claim30, wherein the nucleic acid is encodes bitstream data.
 37. The methodof claim 30, wherein the manipulation is carried out in a region of thenucleic acid that is a barcode.
 38. The method of claim 1, wherein themicrofluidic device is an electrowetting on dielectric (EWOD) device.39. The method of claim 22, wherein the nucleic acid is a barcode. 40.The method of claim 39, wherein the barcode is attached to a nucleicacid memory object.
 41. The method of claim 39, wherein the barcode isnot the exact sequence of the barcode associated to the concept ormetadata, but it mutated away from the barcode by 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 mutations.
 42. The method of claim 41, wherein the mutatedbarcode is associated with metadata or a concept of the nearest barcodeheld in a barcode hash table associating to metadata contained withinthe nucleic acid memory object.
 43. The method of claim 41, wherein themutated barcode is associated with variations of metadata or a conceptof the nearest barcode held in a barcode hash table.
 44. The method ofclaim 39, wherein the barcode is associated with metadata describingbiological information of the nucleic acid sequence contained in thenucleic acid memory object.
 45. The method of claim 44, wherein thenucleic acid sequence is encapsulated within a nucleic acid memoryobject, wherein the nucleic acid memory object encodes a gene, and thebarcode sequence describes one or more features selected from the groupconsisting of gene name, mutations of the gene, the source organism,gene length, the protein(s) encoded the gene, and one or more ligands ofthe encoded protein.
 46. The method of claim 39, wherein the barcode isassociated with metadata describing the digital information contained ina DNA sequence contained in the nucleic acid memory object.
 47. Themethod of claim 46, wherein the nucleic acid sequence encodesinformation about an image or images, and the metadata barcode containsthe amount of any given characteristic in the image, and wherein one ormore point mutations of the barcode of are associated with variedamounts of that characteristic.
 48. The method of claim 47, wherein thecharacteristic of the image is the intensity of one or more colors. 49.The method of claim 46, wherein the DNA sequence encodes a digitalrepresentation of an image or images, and the metadata barcode containsdescriptions of objects in the image or images, wherein the mutations ofthe barcodes of claim 42 are associated with the likeness to the object.